B7 FAMILY MEMBER zB7H6 AND RELATED COMPOSITIONS AND METHODS

ABSTRACT

Disclosed is a newly identified B7 family member, zB7H6, which functions as a counter-receptor for the NK cell triggering receptor, NKp30. Methods and compositions for modulating NKp30-mediated NK cell activity based on the interaction of zB7H6 with NKp30, as well as related screening methods, are also disclosed. Further disclosed are anti-zB7H6 antibodies as well as antibody-drug conjugates comprising an anti-zB7H6 antibody conjugated to a therapeutic agent, including methods for using such antibodies and antibody-drug conjugates to exert therapeutic effects against zB7H6-expressing cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 12/246,214, filed Oct. 6, 2008, which claims the benefit of U.S.Patent Application Ser. No. 60/977,584, filed Oct. 4, 2007, U.S. PatentApplication Ser. No. 61/026,802, filed Feb. 7, 2008, and U.S. PatentApplication Ser. No. 61/095,875, filed Sep. 10, 2008, each of which isincorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION B7 Family

Positive and negative costimulatory signals play critical roles in themodulation of lymphocyte activity, and the molecules that mediate thesesignals have proven to be effective targets for immunomodulatory agents.For example, upon interaction with B7-1 or B7-2 on the surface ofantigen-presenting cells (APC), CD28, the prototypic T cellcostimulatory molecule, emits signals that promote T cell proliferationand differentiation in response to T cell receptor (TcR) engagement,while the CD28 homologue cytotoxic T lymphocyte antigen-4 (CTLA-4)mediates inhibition of T cell proliferation and effector functions. (SeeChambers et al., Ann. Rev. Immunol., 19:565-594, 2001; Egen et al.,Nature Immunol., 3:611-618, 2002.)

Several new molecules with homology to the B7 family have beendiscovered (Abbas et al., Nat. Med., 5:1345-6, 1999; Coyle et al., Nat.Immunol., 2: 203-9, 2001; Carreno et al., Annu. Rev. Immunol., 20:29-53, 2002; Liang et al., Curr. Opin. Immunol., 14: 384-90, 2002), andtheir role in lymphocyte activation is just beginning to be elucidated.These new costimulatory counter-receptors include B7h2, PD-L1, PD-L2,B7-H3 and B7-H4.

The expression of known B7 family members is largely restricted toantigen-presenting cells. Collectively, these studies have revealed thatB7 family members are counter-receptors on lymphoid cells that interactwith cognate receptors on lymphocytes to provide positive or negativecostimulatory signals that play critical roles in the regulation ofcell-mediated immune responses.

Accordingly, there is a need in the art for the identification ofadditional B7 family members, their counter-receptors, and moleculesderived therefrom that have lymphocyte costimulatory activity. This needis based largely on their fundamental biological importance and thetherapeutic potential of agents capable of affecting their activity.Such agents capable of modulating costimulatory signals would findsignificant use in the modulation of immune responses, and are highlydesirable.

NK Cells and NKp30

Natural killer (NK) cells are a subset of lymphocytes active in theimmune system and represent an average of about 15% of mononuclear cellsin human peripheral blood. NK cells were initially describedfunctionally in 1971 by the observation that lethally irradiated micewere capable of rejecting allogeneic or parental strain bone marrow cell(BMC) allografts. (See Cudowicz and Bennett, J. Exp. Med. 134:83-102,1971; Cudowicz and Bennett, J. Exp. Med. 135:1513-1528, 1971.) Cudowiczand Bennett observed that irradiated F1 hybrid H-2-heterozygous mice(A×B) were capable of rejecting parental H-2-homozygous BMC (A or B).This observation conflicted with the classic laws of transplantation inwhich transplantation antigens were thought to inherit co-dominantly andoffspring were obligately tolerant toward parental majorhistocompatability complex (MHC) determinants. (See Cudowicz andBennett, J. Exp. Med. 134:83-102, 1971.) The cells responsible for thisphenomenon were found to be radioresistant and identical to lymphoidcells, which were characterized later in 1975 by their ability tomediate spontaneous killing of tumors in vitro in an MHC-unrestrictedmanner. (See Herberman and Ortaldo, Science, 214:24-30, 1981; Ortaldoand Herberman, Annu. Rev. Immunol. 2:359-394, 1984; Trinchieri, Adv.Immunol. 47:187-376, 1989; Murphy et al., J. Natl. Cancer Inst.85:1475-1482, 1993.) Additional evidence that NK cells alone couldmediate the specificity of marrow graft rejection emerged in 1987 whenit was observed that mice with severe combined immune deficiency (SCID),which cannot develop T and B cells, have normal NK cell function. (SeeMurphy et al., J. Exp. Med. 165:1212-1217, 1987.)

NK cells are currently understood to represent an important arm ofinnate immunity and to play a primary role in immune surveillanceagainst tumors and virally infected cells. Unless activated, however, NKcells are ineffective in performing their normal function, even whenpresent in otherwise sufficient numbers. Indeed, decreased NK cellactivity is associated with cancer and infectious diseases (see Yamazakiet al., Oncology Reports 9:359-363, 2002; Rosenberg et al., CancerResearch 51:5074-5079 (suppl.), 1991; Britteenden et al., Cancer77:1226-1243, 1996; U.S. Pat. Nos. 5,082,833 and 4,883,662). Conversely,as noted above, NK cell activity mediates acute rejection of BMCallografts. Therefore, levels of NK cell activity appear to play animportant role in immune-related disorders.

NK cell activity is typically regulated by the interaction between MHCclass I molecules and inhibitory and activating receptors. (See, e.g.,Barao and Murphy, BB&MT 9:727-741, 2003.) The “missing self” hypothesisis originally based on the observation that tumor cells that lack MHCclass I molecules are susceptible to killing by NK cells. (See Ljunggrenand Karre, Immunol. Today 11:237-244, 1990; Ohlen et al., J. Immunol.145:52-58, 1990.) Investigators additionally observed that human NKcells lyse class-I-deficient Epstein-Barr-virus-transformedB-lymphoblastoid cell lines. (Storkus et al., Proc. Natl. Acad. Sci. USA86:2361-2364, 1989.) Also, it was found that transfection of class Igenes into class I-deficient target cells caused these cells to bepartially or completely resistant to NK cell-mediated lysis. (SeeStorkus et al., supra; Shimizu and DeMars, Eur. J. Immunol., 19:447-451,1989.) MHC class I, however, is not always necessary for protection fromNK-cell-mediated cytotoxicity, and recognition by MHC class I does notalways prevent cytolysis by NK cells. (Barao and Murphy, supra.) Duringrecent years, various MHC-class-I-specific inhibitory and activatingreceptors as well as non-MHC-class-I-specific activating receptors havebeen identified. These receptors are relevant with respect totherapeutic approaches such as, e.g., allogeneic BMT and cancer therapy.(See id.)

Non-MHC-class-I-specific activating receptors, which are capable ofmediating NK cell cytotoxicity against MHC-class-I-deficient or negativetargets, are represented in part by a heterogeneous family of NKcell-specific immunoglobulin-like molecules that are known as naturalcytotoxicity receptors (NCRs). (See, e.g., Moretta et al., Annu. Rev.Immunol. 19:197-223, 2001; Diefenbach and Raulet, Immunol. Rev.,181:170-184, 2001.) In the absence of MHC class I expression (such as,for example, on tumor cells or virus-infected cells), ligation of theseactivating receptors on NK cells triggers target-cell killing. One suchactivating receptor is NKp30, which is selectively and constitutivelyexpressed on mature natural killer (NK) cells and signals through, interalia, coupling with CD3ζ. (See Barao and Murphy, supra.) The target-cellligand to which NKp30 binds has not been previously identified.

This system of innate recognition by NK cells represents a potentiallypowerful tool for clinical application in allogeneic bone marrowtransplantation (BMT), cancer therapy, or treatment of otherNK-cell-associated disorders. (See, e.g., Barao and Murphy, supra.) Forexample, stimulating or inhibiting activation of NKp30 would be usefulfor modulating NK cell activity and treating diseases or disordersassociated with NK cell activity. In particular, enhancement of NK cellactivity by triggering NKp30 would be useful for treatment of diseasesor disorders characterized by insufficient NK cell activity, such ascancer and infectious disease, while inhibition of NK cell activity byblocking NKp30 would be useful for treating NK-cell-mediated disorders,such as, for example, BMC allograft rejection. The present inventionprovides compositions and methods for these and other uses that shouldbe apparent to those skilled in the art from the teachings herein.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides isolated zB7H6polypeptides, including polypeptide fusions, comprising the amino acidsequence of SEQ ID NO:2 or a functional variant or fragment thereof. Forexample, in some embodiments, a zB7H6 polypeptide of the invention is anisolated, soluble polypeptide comprising a polypeptide segment that hasat least 90% or at least 95% sequence identity with the amino acidsequence set forth in residues 25-266 of SEQ ID NO:2, wherein thesoluble zB7H6 polypeptide is capable of specifically binding to humanNKp30. In specific variations, such a soluble zB7H6 polypeptidecomprises a polypeptide segment having the amino acid sequence set forthin residues 25-266 or 1-266 of SEQ ID NO:2. Such soluble polypeptidescan be, for example, soluble fusion proteins. Suitable soluble fusionproteins include polypeptides further comprising an immunoglobulin heavychain constant region (e.g., an Fc fragment), such as an IgG (e.g.,IgG1, IgG2, IgG3, or IgG4), IgM, IgE, IgA, or IgD immunoglobulin heavychain constant region. Other suitable soluble fusion proteins includepolypeptides further comprising a VASP domain.

In another aspect, the present invention provides isolatedpolynucleotides encoding a zB7H6 polypeptide as described herein.Accordingly, in certain embodiments, the present invention provides anisolated polynucleotide comprising a polynucleotide segment encoding asoluble zB7H6 polypeptide, the zB7H6 polypeptide comprising apolypeptide segment that has at least 90% sequence identity with theamino acid sequence set forth in residues 25-266 of SEQ ID NO:2, andwherein the zB7H6 polypeptide is capable of specifically binding tohuman NKp30. In a specific variation, the encoded soluble zB7H6polypeptide comprises a polypeptide segment having the amino acidsequence set forth in residues 25-266 of SEQ ID NO:2. The encodedsoluble polypeptide can be, for example, a soluble fusion protein, suchas a soluble fusion protein comprising an immunoglobulin heavy chainconstant region or a VASP domain. In certain variations, thepolynucleotide segment encoding the zB7H6 polypeptide comprisesnucleotides 73-798 or 1-798 of SEQ ID NO:1.

In yet other aspects, the present invention provides vectors, includingexpression vectors, comprising a polynucleotide as above. For example,in some embodiments, the present invention provides an expression vectorcomprising the following operably linked elements: a transcriptioninitiation region; a DNA segment encoding a soluble zB7H6 polypeptide,the zB7H6 polypeptide comprising a polypeptide segment that has at least90% sequence identity with the amino acid sequence set forth in residues25-266 of SEQ ID NO:2, wherein the zB7H6 polypeptide is capable ofspecifically binding to human NKp30; and a transcription terminationregion. In other, related aspects, the present invention provides hostcells comprising such vectors, as well as methods for producing a zB7H6polypeptide. In some embodiments, a method of producing a soluble zB7H6polypeptide includes culturing a host cell comprising an expressionvector as above under conditions in which the polypeptide is expressed,and recovering the expressed polypeptide.

The present invention also provides isolated antibodies thatspecifically bind to a zB7H6 polypeptide as described herein. Forexample, in certain embodiments, the present invention provides anantibody that specifically binds to a polypeptide segment having theamino acid sequence set forth in residues 25-266 of SEQ ID NO:2. In somesuch variations, the antibody inhibits the interaction of zB7H6 withhuman NKp30. Particularly suitable antibodies are monoclonal antibodies,such as, e.g., human or humanized monoclonal antibodies. Anti-zB7H6antibodies also include single chain antibodies.

In still another aspect, the present invention provides methods formodulating human natural killer (NK) cell activity. Some such methodsinclude enhancing NK cell activity by contacting a human NK cell with acell expressing a recombinant, membrane-bound zB7H6 polypeptide, thezB7H6 polypeptide comprising a polypeptide segment that has at least 90%sequence identity with the amino acid sequence set forth in residues25-266 of SEQ ID NO:2, and wherein the zB7H6 polypeptide is capable ofspecifically binding to human NKp30. In a specific variation, the zB7H6polypeptide segment has the amino acid sequence set forth in residues25-266 of SEQ ID NO:2.

Other methods for modulating NK cell activity include, e.g., decreasingNK cell activity against a zB7H6-expressing cell. Such methods generallycomprise contacting a cell expressing functional zB7H6, in the presenceof a human NK cell, with an effective amount of an antibody thatspecifically binds to a polypeptide segment having the amino acidsequence set forth in residues 25-266 of SEQ ID NO:2, wherein theantibody inhibits the interaction of zB7H6 with human NKp30. Suchmethods for decreasing NK cell activity are useful, for example, in thetreatment of bone marrow cell (BMC) allograft rejection. Accordingly, incertain variations, a method of the invention includes treating bonemarrow cell (BMC) allograft rejection in a human subject byadministering to the human subject, in an amount effective to inhibit NKcell activity and thereby treat the acute BMC allograft rejection, anantibody that (a) specifically binds to a polypeptide segment having theamino acid sequence set forth in residues 25-266 of SEQ ID NO:2 and (b)inhibits the interaction of zB7H6 with human NKp30. Particularlysuitable antibodies include monoclonal antibodies (e.g., human orhumanized monoclonal antibodies). Antibodies for treating BMC can alsobe single chain antibodies.

In another aspect, the present invention provides methods for inducingantibody dependent cellular cytotoxicity (ADCC) against azB7H6-expressing cell. Such methods generally include contacting thezB7H6-expressing cell with an effective amount an antibody thatspecifically binds to a polypeptide segment having the amino acidsequence set forth in residues 25-266 of SEQ ID NO:2, wherein thecontacting is in the presence of an NK cell or a CD8⁺ T cell expressingan Fc receptor having ADCC activity, and wherein the antibody comprisesan Fc region capable of binding the Fc receptor. Suitable anti-zB7H6antibodies include monoclonal antibodies, including, for example, humanor humanized monoclonal antibodies, as well as single chain antibodies.In certain variations, the Fc region is a single chain Fc (scFc). ThezB7H6-expressing cell can be, for example, a zB7H6-expressing cancercell. zB7H6 cancer cells particularly amenable to targeted killing usingthese methods include, e.g., colon cancer cells, liver cancer cells,cervical cancer cells, lung cancer cells, pancreatic cancer cells,prostate cancer cells, prohemocytic leukemia cells, B-cell lymphomacells, monocytic lymphoma cells, erythroleukemia cells, Burkitt'slymphoma cells, and chronic myelogenous leukemia cells.

In yet another aspect, the present invention provides methods forinducing complement dependent cytotoxicity (CDC) against azB7H6-expressing cell. Such methods generally include contacting thezB7H6-expressing cell with an effective amount an antibody thatspecifically binds to a polypeptide segment having the amino acidsequence set forth in residues 25-266 of SEQ ID NO:2, wherein thecontacting is in the presence of complement, and wherein the anti-zB7H6antibody comprises an Fc region having CDC activity. Suitable anti-zB7H6antibodies include monoclonal antibodies, including, for example, humanor humanized monoclonal antibodies, as well as single chain antibodies.In certain variations, the Fc region is a single chain Fc (scFc). ThezB7H6-expressing cell can be, for example, a zB7H6-expressing cancercell. zB7H6 cancer cells particularly amenable to targeted killing usingthese methods include, e.g., colon cancer cells, liver cancer cells,cervical cancer cells, lung cancer cells, pancreatic cancer cells,prostate cancer cells, prohemocytic leukemia cells, B-cell lymphomacells, monocytic lymphoma cells, erythroleukemia cells, Burkitt'slymphoma cells, and chronic myelogenous leukemia cells.

In another, related aspect, the present invention provides methods fortreating a zB7H6-expressing cancer in a subject. Such methods generallyinclude administering to the subject an effective amount of an antibodythat specifically binds to a polypeptide segment having the amino acidsequence set forth in residues 25-266 of SEQ ID NO:2, wherein theantibody comprises an Fc region having ADCC and/or CDC activity.Suitable anti-zB7H6 antibodies include monoclonal antibodies, including,for example, human or humanized monoclonal antibodies, as well as singlechain antibodies. In certain variations, the Fc region is a single chainFc (scFc). zB7H6-expressing cancers particularly amenable to treatmentusing such methods include, for example, cancers of the colon, liver,cervix, lung, pancreas, and prostate, as well as cancers of the bloodsuch as, e.g., prohemocytic leukemia, B-cell lymphoma, monocyticlymphoma, erythroleukemia, Burkitt's lymphoma, or chronic myelogenousleukemia.

In another aspect, the present invention provides an antibody-drugconjugate comprising an antibody that specifically binds to apolypeptide segment having the amino acid sequence set forth in residues25-266 of SEQ ID NO:2, wherein the antibody is conjugated to a cytotoxicagent. In certain embodiments, the antibody that binds the amino acidsequence of residues 25-266 of SEQ ID NO:2 is a monoclonal antibody suchas, for example, a human or humanized monoclonal antibody. In othervariations, the antibody is a single chain antibody. Suitable cytotoxicagents include, for example, anti-tubulin agents, DNA minor groovebinding agents, DNA minor groove alkylating agents, duocarmycins, andpuromycins. Particularly suitable anti-tubulin agents include, e.g.,dolastatins, vinca alkaloids, podophyllatoxins, taxanes, baccatinderivatives, cryptophysins, maytansinoids, and combretastatins.

In typical embodiments of an antibody-drug conjugate as summarize above,the antibody is conjugated to the cytotoxic agent via a linker.Particularly suitable linkers are linker that are cleavable underintracellular conditions, such as, for example, a peptide linkercleavable by an intracellular protease (e.g., cleavable by a lysosomalprotease or an endosomal protease). Linkers cleavable underintracellular conditions may include dipeptide linkers, such as, forexample, a val-cit linker or a phe-lys linker. In other variations, thecleavable linker is hydrolyzable at a pH of less than 5.5 (e.g., ahydrazone linker). In yet other variations, the cleavable linker is adisulfide linker.

The present invention further includes pharmaceutical compositioncomprising an antibody-drug conjugate as above and at least onepharmaceutically acceptable carrier.

In yet another aspect, the present invention provides a method fordepleting or inhibiting the growth of zB7H6-expressing cells within acell population comprising said zB7H6-expressing cells. Generally, themethod includes contacting said zB7H6-expressing cells with an effectiveamount of an antibody-drug conjugate as above. In certain embodiments,the method is used in vivo to treat a zB7H6-expressing cancer in asubject by administering to the subject an effective amount of theantibody-drug conjugate. In particular variations, the zB7H6-expressingcancer is a cancer of the colon, liver, cervix, lung, pancreas, orprostate. In yet other variations, the zB7H6-expressing cancer is aprohemocytic leukemia, a B-cell lymphoma, a monocytic lymphoma, aerythroleukemia, Burkitt's lymphoma, or a chronic myelogenous leukemia.

The present invention further provides methods of screening for anantagonist or an agonist of the interaction of zB7H6 with NKp30. Forexample, in certain embodiments, a method of screening for antagonist ofthe interaction of zB7H6 with NKp30 generally includes (a) contacting anagent with a zB7H6 polypeptide in the presence of an NKp30 polypeptide;(b) detecting a measure of the interaction of the zB7H6 polypeptide withthe NKp30 polypeptide; and (c) determining whether the level of thezB7H6/NKp30 interaction measured in step (b) is significantly lessrelative to the level of interaction measured for control zB7H6 andNKp30 polypeptides in the absence of the agent, such that if the levelof zB7H6/NKp30 interaction is less, then the agent is identified as anantagonist of the interaction of zB7H6 with NKp30. In other embodiments,a method of screening an agent for an agonist of the interaction ofzB7H6 with NKp30 generally includes (a) contacting an agent with a zB7H6polypeptide in the presence of an NKp30 polypeptide; (b) detecting ameasure of the interaction of the zB7H6 polypeptide with the NKp30polypeptide; and (c) determining whether the level of the zB7H6/NKp30interaction measured in step (b) is significantly greater relative tothe level of interaction measured for control zB7H6 and NKp30polypeptides in the absence of the agent, such that if the level ofzB7H6/NKp30 interaction is greater, then the agent is identified as anagonist of the interaction of zB7H6 with NKp30.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict inhibition of NK-92 cytolytic activity againstK562 targets with a soluble NKp30 fusion protein. In particular, solubleNKp30/VASP A1683F inhibited the cytolytic activity of NK-92 cellsagainst K562 targets. (See FIG. 1A.) In a separate cytolytic assayexperiment using different concentrations of soluble NKp30/VASP (0.25,0.5, 1.0, 2.0, 4.0, 8.0, and 16.0 μg/ml) added to wells containing NK-92effectors and K562 targets at an effector:target ratio of 9:1, solubleNKp30 inhibited lysis by NK-92 cells in a dose dependent manner. (SeeFIG. 1B.)

FIG. 2 depicts binding of soluble NKp30 fusion protein to K562 cells.K562 cells were incubated in the presence of NKp30/mFc2 fusion proteinfollowed by secondary labeling with PE anti-mIgG and analyzed for PEstaining by FACS. NKp30/mFc2 bound to K562 cells (“No Comp.”). Thisbinding was competable with a second soluble NKp30 fusion protein,NKp30/VASP, but not competable with control VASP proteins (“hzB7R1/Vasp”and “B7-DC/Vasp”).

FIG. 3 depicts binding of soluble NKp30 fusion protein to K562 cells,but not to BaF3 cells. K562 cells and BaF3 cells were probed withNKp30/mFc2 conjugated to biotin, followed by secondary labeling withPE-conjugated streptavidin.

FIGS. 4 and 5A-5B depict crosslinking of K562 cells and biotinylatedNKp30/mFc2. Four samples, the sample of interest and three negativecontrol samples, were analyzed. The sample of interest was K562 cellsincubated with biotinylated NKp30/mFc2. The three negative controlsamples were K562 cells with no NKp30 and BaF3 cells with and withoutNKp30. Each sample was reacted with a chemical crosslinker to covalentlylink any protein-protein interactions and the biotinylated componentswere separated and collected by streptavidin agarose precipitation.Samples were split and run on identical 4-12% Nu-Page gels. One gel wasused for a Western blot probed with streptavidin-HRP (see FIGS. 4 and5B). The second gel was coomassie-stained (see FIG. 5A). FIGS. 5A and 5Bshow the coomassie-stained gel and corresponding Western blotjuxtaposed.

FIG. 6 depicts the amino acid sequence of protein DKFZP686I21167(subsequently designated zB7H6), with peptides identified by LC-MS/MSunderlined in bold.

FIG. 7 depicts the gene structure profile of protein DKFZP686I21167(subsequently designated zB7H6). The gene structure profile isSignal-2-IgV-2-IgC-2-TMD-0-LgEx, where the integers “2” and “0” denotethe phasing between exons 1 through 5 coding for, respectively, a leadersequence (“S”), an IgV domain, an IgC domain, a transmembrane domain(“TMD”), and an intracellular domain with homology to Gag polyprotein.“SxYxxL,” “YxxQ,” and “PxxPxxP” denote potential signaling motifs withinthe intracellular domain of zB7H6 (respectively, an ITIM motif, an SH2binding motif, and an SH3 binding motif).

FIG. 8 depicts binding of soluble NKp30 to BaF3 cells expressingfull-length zB7H6. Soluble NKp30/VASP-A647 bound to cells electroporatedwith the human zB7H6 expression vector (see FIGS. 8A and 8B—solid,unfilled line), but not to control cells containing an empty vectorcontrol (see FIGS. 8A and 8B—filled line). Staining with NKp30/VASP-A647was not observed in the presence of a 100-fold excess of unlabeledNKp30VASP (see FIG. 8A—dashed line), but was observed in the presence ofa 100-fold excess of unlabeled irrelevant VASP protein (see FIG.8B—dashed line).

FIG. 9 depicts NK-92 lysis of P815 cells. NK-92 cells were cultured withP815 cells at an effector:target ratio from 27:1 down to 1:1 in 3-folddilutions. NK-92 cells did not lyse wild-type P815 cells or P815 cellstransfected with two non-triggering control proteins (hIgSF1 and hB7H1),while addition of an activating anti-NKp30 monoclonal antibody triggeredre-directed lysis. Transfection of either hCD86 or zB7H6 triggereddirect killing of P815 cells.

FIG. 10 depicts inhibition of NK-92 cytolytic activity against zB7H6expressing cells with soluble NKp30 and anti-zB7H6 antibody. NK-92 cellswere cultured with either K562 cells or P815 cells expressing zB7H6 atan effector:target ratio of 9 to 1. A soluble form of NKp30(NKp30/VASP), a control VASP protein (B7H3/VASP), an anti-zB7H6polyclonal antibody, and an irrelevant control antibody were added tosome wells. Soluble NKp30/VASP and anti-zB7H6 polyclonal antibodyinhibited the cytolytic activity of NK-92 cells against K562 and P815zB7H6 targets, while the VASP and antibody controls had no effect.

FIGS. 11A-11C depict specific binding of soluble NKp30 to K562, P815zB7H6 and 293F cells. K562, P815 zB7H6 and 293F cells were probed byFACS with a biotinylated NKp30/mFc2, either in the absence or presenceof a 100-fold mass excess of NKp30VASP, zB7H6/VASP, or a control VASPprotein (B7H3/VASP). Following incubation with biotinylated NKp30/mFc2,cells were washed and stained with streptavidin-PE. Cells were thenwashed and analyzed for PE staining on a FACSCalibur. NKp30/mFc2-biotinbound to K562 (11A), 293F (11B), and P815 zB7H6 (11C) cells (“NoCompetition”). This binding was competable with NKp30/VASP andzB7H6/VASP, but not with control VASP protein (“B7H3/VASP”).

FIGS. 12A-12D depict specific binding of anti-B7H6 antibody to K562,P815 zB7H6, and 293F cells. K562, P815, P815 zB7H6 and 293F cells wereprobed with an A647 conjugated form of anti-zB7H6 mouse polyclonalantibody (E10607). Cells were incubated with whole human IgG to block Fcreceptors, and A647-conjugated anti-zB7H6 (“anti-zB7H6-A647”) antibodywas added to cells in the absence or presence of a 100-fold mass excessof a VASP protein (zB7H6/VASP or a control VASP protein, B7H3/VASP).Following incubation with antibody, cells were washed and analyzed forAPC staining on a FACSCalibur. Anti-zB7H6 bound to K562 (12B), P815zB7H6 (12C), and 293F (12D) cells but not to untransfected P815 cells(12A) (“No Competition”). This binding was competable with zB7H6/VASP,but not with control VASP protein.

FIGS. 13A-13C illustrate the amino acid sequences of certainimmunoglobulin Fc polypeptides. Amino acid sequence numbers are based onthe EU index (Kabat et al., Sequences of Proteins of ImmunologicalInterest, US Department of Health and Human Services, NIH, Bethesda,1991). The illustrated sequences include a wild-type human sequence(“wt”; SEQ ID NO:29) and five variant sequences, designated Fc-488 (SEQID NO:30), Fc4 (SEQ ID NO:31), Fc5 (SEQ ID NO:32), Fc6 (SEQ ID NO:33),and Fc7 (SEQ ID NO:34). The Cys residues normally involved in disulfidebonding to the light chain constant region (LC) and heavy chain constantregion (HC) are indicated. A “.” indicates identity to wild-type at thatposition. *** indicates the stop codon; the C-terminal Lys residue hasbeen removed from Fc6. Boundaries of the hinge, C_(H)2, and C_(H)3domains are shown.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

The present invention is directed to the identification andcharacterization of zB7H6, a novel member of the B7 family of cellularreceptors, and the discovery of its ability to bind to NKp30. The novelreceptor of the present invention is denominated “zB7H6” and is distinctfrom previously known members of the B7 family such as B7-1, B7-2, B7h2,PD-L1, PD-L2, B7-H3 and B7-H4. Methods and compositions for modulatingzB7H6-mediated signaling such as, e.g., modulating the naturalinteraction of zB7H6 and NKp30 are also provided, having multipletherapeutic applications for immunotherapy, including immunotherapy for,e.g., cancer and infectious disease.

An illustrative nucleotide sequence that encodes human zB7H6 is providedby SEQ ID NO:1; the encoded polypeptide is shown in SEQ ID NO:2. ThezB7H6 polypeptide of SEQ ID NO:2 comprises an extracellular domain ofapproximately 242 amino acid residues (residues 25-266 of SEQ ID NO:2),a transmembrane domain of approximately 18 amino acid residues (residues267-284 of SEQ ID NO:2), and an intracellular domain of approximately158 amino acid residues (residues 285-454 of SEQ ID NO:2). zB7H6 alsohas an IgV domain of approximately 117 amino acid residues (residues25-141 of SEQ ID NO:2) and an IgC domain of approximately 97 amino acidresidues (residues 142-238 of SEQ ID NO:2). There are also severalpotential signaling motifs within the intracellular domain of zB7H6,including an ITIM motif (SaYtpL, amino acid residues 293-298 of SEQ IDNO:2); an SH2 binding motif (YqlQ, amino acid residues 229-332 of SEQ IDNO:2); and an SH3 binding motif (PdaPilPvsP, amino acid residues 418-427of SEQ ID NO:2).

zB7H6 was identified as a member of the B7 family of cellular receptorsbased on B7 family gene profiling. The gene structure profile isSignal-2-IgV-2-IgC-2-TMD-0-LgEx. (See FIG. 7.) The extracellular regionof this profile matches a B7 gene structure model, which includescharacteristic exon patterns in which the first exon encodes a leadersequence, the second exon encodes an IgV domain and the third exonencodes an IgC domain. Another characteristic feature of the B7 familygene structure is the phasing of the exons: in the region correspondingto the extracellular domain, B7 family members show a conserved phasingof 2 between exons 1 to 4. (See id.)

zB7H6 was identified as a counter-receptor for NKp30, a receptorselectively expressed on mature natural killer (NK) cells and which isinvolved in human natural cytotoxicity as an activatory receptor. NKcells are typically prevented from attacking normal tissue by theinteraction between MHC class I molecules and inhibitory receptors. Inthe absence, however, of MHC class I expression (such as, for example,on tumor cells or virus-infected cells), ligation of activatingreceptors on NK cells triggers target-cell killing. Such triggeringNK-cell receptors include NKp30, NKp44, NKp46, NKG2D, and DNAM1. Theactivating target-cell ligand to which NKp30 binds had not beenpreviously identified, and the identification of zB7H6 as thecounter-receptor for NKp30 enables a variety of therapeutic agentscapable of mimicking or interfering with the interaction of zB7H6 andNKp30 to modulate NK lymphocyte activity for the purpose of treating,among other conditions, cancer, infectious disease, or NK-cell mediatedallograft rejection. For example, a reagent that mimics the zB7H6-NKp30interaction, including a soluble form of zB7H6 comprising theextracellular domain, can be used to facilitate NK cell responses to atumor or virus-infected cells by activating the NKp30 stimulatorysignal. Conversely, an agent that blocks the zB7H6-NKp30 interaction,such as, for example, an anti-zB7H6 antibody that competes for bindingwith NKp30, can be used to inhibit NK cell-mediated responses such as,for example, in acute bone marrow cell (BMC) allograft rejection.

Accordingly, in one aspect, the present invention provides zB7H6polypeptides that are useful in the modulation of NK cell activity andin the treatment of disorders such as cancer, infectious disease, or NKcell-mediated allograft rejection. Generally, such zB7H6 polypeptidescomprise the zB7H6 extracellular domain (residues 25-266 of SEQ IDNO:2); a functional variant of the zB7H6 extracellular domain having atleast 80% (e.g., at least 90% or at least 95%) identity with residues25-266 of SEQ ID NO:2 and capable of binding to NKp30; or a functionalfragment of the aforementioned zB7H6 extracellular domain or domainvariant, which fragment is capable of binding to NKp30. In somevariations, the zB7H6 polypeptide has the amino acid sequence ofresidues 25-454 of SEQ ID NO:2 (e.g., the polypeptide of SEQ ID NO:2),or a functional variant of zB7H6 having at least 80% (e.g., at least90%, or at least 95%) identity with residues 25-454 of SEQ ID NO:2. Incertain embodiments, the zB7H6 polypeptide is a soluble zB7H6polypeptide lacking a functional transmembrane domain. Particularlysuitable soluble zB7H6 polypeptides include fusion proteins comprisingor consisting of the zB7H6 extracellular domain, or the functionalvariant or fragment thereof, and a heterologous polypeptide. In somesuch variations, the heterologus polypeptide is an immunoglobulinmoiety; a particularly suitable immunoglobulin moiety is animmunoglobulin heavy chain constant region, such as a human F_(c)fragment. In other variations, the heterologous polypeptide is avasodialator-stimulated phosphoprotein (VASP) domain, which isparticularly suitable for preparation of multimeric (e.g., tetrameric)forms of soluble zB7H6. In some embodiments, the soluble fusion proteinfurther includes a polypeptide linker.

The present invention also provides polynucleotides, including vectors,encoding soluble zB7H6 polypeptides of the invention, as well as hostcells comprising such polynucleotides. In some aspects of the invention,such polynucleotides, vectors, and host cells are used in methods forpreparing a soluble zB7H6 protein. Such methods generally includeculturing a host cell transformed or transfected with an expressionvectors encoding the soluble zB7H6 protein under conditions in which theprotein is expressed, and recovering the soluble zB7H6 protein from thehost cell.

The present invention further provides antibodies that specifically bindto the extracellular domain of zB7H6. In various embodiments, suchantibodies bind to monomeric and/or multimeric forms of zB7H6,including, for example, to monomeric or multimeric forms of solublezB7H6. Such antibodies include agonist antibodies, neutralizingantibodies, polyclonal antibodies, murine monoclonal antibodies,humanized antibodies derived from murine monoclonal antibodies, humanmonoclonal antibodies, and antigen-binding fragments thereof.Illustrative antibody fragments include F(ab′)₂, F(ab)₂, Fab′, Fab, Fv,scFv, and minimal recognition units. Neutralizing antibodies bind zB7H6such that its interaction with NKp30 is inhibited or blocked.

The present invention further includes pharmaceutical compositionscomprising a pharmaceutically acceptable carrier and a soluble zB7H6polypeptide or anti-zB7H6 antibody as described herein. Suchcompositions can be used in therapeutic methods according to the presentinvention.

In other aspects, the present invention provides methods for modulatingNK cell activity using agents that either mimic or block zB7H6 activity.Suitable agents that mimic zB7H6 activity include soluble forms of zB7H6comprising the extracellular zB7H6 domain, or functional variants orfragments thereof capable of binding to and stimulating NKp30 activity.Alternative agonists include gene therapy vectors capable ofrecombinantly producing functional zB7H6 molecules intracellularly,small molecule enhancers of zB7H6 expression and/or zB7H6-mediatedsignaling, and the like. Suitable zB7H6 blocking agents includeanti-zB7H6 antibodies capable of binding to at least a portion of theextracellular domain of zB7H6 and interfering with the interaction ofzB7H6 with NKp30; small molecule inhibitors of the zB7H6 interactionwith NKp30, and the like. Alternative zB7H6 antagonists further includeantisense oligonucleotides directed to the zB7H6 nucleic acid sequence,inhibitory RNA sequences, small molecule inhibitors of B7H6 expressionand/or intracellular signaling, and the like.

For example, in some embodiments, the present invention provides amethod for treating a disease or disorder characterized by insufficientnatural killer (NK) cell activity (e.g., a cancer or an infectiousdisease) by administering to a subject an effective amount of a solublezB7H6 polypeptide. In other aspects, the present invention provides amethod for decreasing human natural killer (NK) cell activity against azB7H6-expressing cell by contacting the zB7H6-expressing cell, in thepresence of a human NK cell, with an effective amount of an antibodythat specifically binds to the extracellular domain of zB7H6 and thatinhibits the interaction of zB7H6 with human NKp30; such methods can beused, for example, in vivo for treating NK-cell-mediated allograftrejection, particularly acute BMC allograft rejection.

These and other aspects of the invention will become evident uponreference to the following detailed description. In addition, variousreferences are identified below and are incorporated by reference intheir entirety.

II. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art pertinent to the methods and compositions described. As usedherein, the following terms and phrases have the meanings ascribed tothem unless specified otherwise.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol. 7:551, 1993), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol. 1:47, 1990), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938, 1992), AP2 (Ye etal., J. Biol. Chem. 269:25728, 1994), SP1, cAMP response element bindingprotein (CREB; Loeken, Gene Expr. 3:253, 1993) and octamer factors (seegenerally Watson et al., eds., Molecular Biology of the Gene, 4th ed.(The Benjamin/Cummings Publishing Company, Inc. 1987), and Lemaigre andRousseau, Biochem. J. 303:1, 1994). If a promoter is an induciblepromoter, then the rate of transcription increases in response to aninducing agent. In contrast, the rate of transcription is not regulatedby an inducing agent if the promoter is a constitutive promoter.Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

In the context of host cell expression of DNA, a “heterologous” peptideor polypeptide is a peptide or polypeptide encoded by a non-host DNAmolecule, i.e., a peptide or polypeptide encoded by a heterologous DNAmolecule.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, that has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that produces zB7H6from an expression vector. In contrast, zB7H6 can be produced by a cellthat is a “natural source” of zB7H6, and that lacks an expressionvector.

“Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

A “fusion protein” is a hybrid protein comprising at least twopolypeptide segments that are, relative to each other, derived fromdifferent proteins. In this context, “different proteins” means thateach protein corresponds to a different gene locus. A proteincorresponds to a gene locus if it is encoded by an allele correspondingto the gene locus, or if the protein has at least 80% sequence identityto a protein encoded by such an allele. Polypeptide segments derivedfrom different proteins are also referred to herein as being“heterologous” with respect to each other. Thus, for example, in thecontext of a fusion protein comprising a zB7H6 polypeptide segment(e.g., the extracellular domain, or a functional variant or fragmentthereof) and a second polypeptide segment from a protein different thanzB7H6, the second polypeptide segment is also referred to herein asbeing a “heterologous polypeptide segment” or “heterologouspolypeptide.” Such heterologous polypeptides include, for example,immunoglobulin constant regions and VASP domains, as further describedherein.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “counter-receptor.” This interactionmediates the effect of the counter-receptor on the cell. Receptors canbe membrane bound, cytosolic or nuclear; monomeric (e.g., thyroidstimulating hormone receptor, beta-adrenergic receptor) or multimeric(e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSFreceptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).Membrane-bound receptors are characterized by a multi-domain structurecomprising an extracellular counter-receptor-binding domain and anintracellular effector domain that is typically involved in signaltransduction. In certain membrane-bound receptors, the extracellularcounter-receptor-binding domain and the intracellular effector domainare located in separate polypeptides that comprise the completefunctional receptor.

In general, the binding of counter-receptor to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-counter-receptor interactions includegene transcription, phosphorylation, dephosphorylation, increases incyclic AMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

A “soluble receptor” is a receptor polypeptide that is not bound to acell membrane. Soluble receptors are most commonlycounter-receptor-binding polypeptides that lack transmembrane andcytoplasmic domains, and other linkage to the cell membrane such as viaglycophosphoinositol (GPI). Soluble receptors can comprise additionalamino acid residues, such as affinity tags that provide for purificationof the polypeptide or provide sites for attachment of the polypeptide toa substrate, or immunoglobulin constant region sequences. Manycell-surface receptors have naturally occurring, soluble counterpartsthat are produced by proteolysis or translated from alternativelyspliced mRNAs. Soluble receptors can be monomeric, homodimeric,heterodimeric, or multimeric, with multimeric receptors generally notcomprising more than 9 subunits, preferably not comprising more than 6subunits, and most preferably not comprising more than 3 subunits.Receptor polypeptides are said to be substantially free of transmembraneand intracellular polypeptide segments when they lack sufficientportions of these segments to provide membrane anchoring or signaltransduction, respectively. For example, representative solublereceptors for zB7H6 include, for instance the soluble receptor as shownin SEQ ID NO:17 or 19.

The term “secretory signal sequence” denotes a DNA sequence that encodesa peptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure,such as 96%, 97%, or 98% or more pure, or greater than 99% pure. One wayto show that a particular protein preparation contains an isolatedpolypeptide is by the appearance of a single band following sodiumdodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the proteinpreparation and Coomassie Brilliant Blue staining of the gel. However,the term “isolated” does not exclude the presence of the samepolypeptide in alternative physical forms, such as dimers oralternatively glycosylated or derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a polypeptide encoded by asplice variant of an mRNA transcribed from a gene.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, co-stimulatory molecules, hematopoieticfactors, and the like, and synthetic analogs of these molecules.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/counter-receptor pairs, antibody/antigen (or hapten or epitope)pairs, sense/antisense polynucleotide pairs, and the like. Wheresubsequent dissociation of the complement/anti-complement pair isdesirable, the complement/anti-complement pair preferably has a bindingaffinity of less than 10⁹ M⁻¹.

The term “antibody,” as used herein, refers to immunoglobulinpolypeptides and immunologically active portions of immunoglobulinpolypeptides, i.e., polypeptides of the immunoglobulin family, orfragments thereof, that contain an antigen binding site thatimmunospecifically binds to a specific antigen (e.g., the extracellulardomain of zB7H6).

An “anti-idiotype antibody” is an antibody that binds with the variableregion domain of an immunoglobulin. In the present context, ananti-idiotype antibody binds with the variable region of an anti-zB7H6antibody, and thus, an anti-idiotype antibody mimics an epitope ofzB7H6.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody. For example, an anti-zB7H6 monoclonal antibody fragment bindsto an epitope of zB7H6.

The term “antibody” also encompasses genetically engineered intactantibodies or fragments such as, for example, chimeric antibodies,humanized antibodies, “Fv” fragments consisting of the variable regionsof the heavy and light chains, polypeptides consisting of the lightchain variable region, recombinant single chain antibodies in whichlight and heavy variable regions are connected by a peptide linker(“scFv proteins”), minimal recognition units consisting of the aminoacid residues that mimic the hypervariable region, and the like, as wellas synthetic antigen-binding peptides and polypeptides.

A “chimeric antibody” is a recombinant protein that contains thevariable domains and complementary determining regions derived from arodent antibody, while the remainder of the antibody molecule is derivedfrom a human antibody.

“Humanized antibodies” are recombinant proteins in which murinecomplementarity determining regions of a monoclonal antibody have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain. Construction of humanizedantibodies for therapeutic use in humans that are derived from murineantibodies, such as those that bind to or neutralize a human protein, iswithin the skill of one in the art.

The terms “Fc fragment,” “Fc region,” or “Fc domain,” as used herein,are synonymous and refer to the portion of an antibody that isresponsible for binding to antibody receptors on cells and the C1qcomponent of complement. Fc stands for “fragment crystalline,” thefragment of an antibody that will readily form a protein crystal.Distinct protein fragments, which were originally described byproteolytic digestion, can define the overall general structure of animmunoglobulin protein. As originally defined in the literature, the Fcfragment consists of the disulfide-linked heavy chain hinge regions,C_(H)2, and C_(H)3 domains. However, more recently the term has beenapplied to a single chain consisting of C_(H)3, C_(H)2, and at least aportion of the hinge sufficient to form a disulfide-linked dimer with asecond such chain. For a review of immunoglobulin structure andfunction, see Putnam, The Plasma Proteins, Vol. V (Academic Press, Inc.,1987), pp. 49-140; and Padlan, Mol. Immunol. 31:169-217, 1994. As usedherein, the term Fc includes variants of naturally occurring sequences.

The terms “single chain Fc,” “single chain Fc domain,” and “scFc,” asused herein, are synonymous and refer to a polypeptide fusion comprisingtwo Fc domain monomers joined by a flexible linker, such that the two Fcmonomers are capable of dimerization to form a functional, dimeric Fcdomain capable of binding Fc receptors. Single chain Fc polypeptides arefurther described in International PCT Patent Application No.US08/060,852, entitled “Single Chain Fc, Methods of Making, and Methodsof Treatment,” filed Apr. 18, 2008, the disclosure of which isincorporated by reference herein in its entirety.

The term “Fc region having ADCC activity,” as used herein, refers to anFc domain capable of mediating antibody dependent cellular cytotoxicity(ADCC) through binding of a cytolytic Fc receptor (e.g., FcγRIIIα) on acytolytic immune effector cell expressing the Fc receptor (e.g., an NKcell or CD8⁺ T cell).

The term “complement” refers collectively to those components in normalserum that, together with antigen-bound antibodies, exhibit the abilityto lyse cells. Complement consists of a group of serum proteins that actin concert and in an orderly sequence to exert their effect.

The terms “classical complement pathway” and “classical complementsystem,” as used herein, are synonymous and refer to a particularpathway for the activation of complement. The classical pathway requiresantigen-antibody complexes for initiation and involves the activation,in an orderly fashion, of nine major protein components designated C1through C9. For several steps in the activation process, the product isan enzyme that catalyzes the subsequent step. This cascade providesamplification and activation of large amounts of complement by arelatively small initial signal.

The term “Fc region having CDC activity,” as used herein, refers to anFc domain capable of mediating complement dependent cytotoxicity (CDC)through binding of C1q complement protein and activation of theclassical complement system.

The term “agent” as used herein means an element, compound, or othermolecular entity, including, e.g., a pharmaceutical, therapeutic, orpharmacologic compound. Agents can be natural or synthetic or acombination thereof. A “therapeutic agent” is an agent that exerts atherapeutic (e.g., beneficial) effect on a cell or a tissue (e.g., on acell or tissue expressing zB7H6, such as a zB7H6-expressing cancercell), either alone or in combination with another agent (e.g., aprodrug converting enzyme in combination with a prodrug). In certainaspects of the present invention, a “therapeutic agent” is an agentconjugated to an antibody to produce a conjugate that is useful fortherapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes. In some variations, a therapeutic agent forconjugation to an antibody is an agent that exerts a cytotoxic orcytostatic effect.

“Cytotoxic effect,” in reference to the effect of an agent on a cell,means killing of the cell. “Cytostatic effect” means an inhibition ofcell proliferation. A “cytotoxic agent” means an agent that has acytotoxic or cytostatic effect on a cell, thereby depleting orinhibiting the growth of, respectively, cells within a cell population.

A “detectable label” is a molecule or atom which can be conjugated to anantibody moiety to produce a molecule useful for diagnosis. Examples ofdetectable labels include chelators, photoactive agents, radioisotopes,fluorescent agents, paramagnetic ions, or other marker moieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952, 1985),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See generally Ford et al., Protein Expression and Purification2:95, 1991. DNA molecules encoding affinity tags are available fromcommercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

A “naked antibody” is an entire antibody, as opposed to an antibodyfragment, which is not conjugated with a therapeutic agent. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric and humanizedantibodies.

The term “monoclonal antibody” refers to an antibody that is derivedfrom a single cell clone, including any eukaryotic or prokaryotic cellclone, or a phage clone, and not the method by which it is produced.Thus, the term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology.

As used herein, the term “antibody component” includes both an entireantibody and an antibody fragment.

An “immunoconjugate” is a conjugate of an antibody component with atherapeutic agent or a detectable label.

As used herein, the term “antibody fusion protein” refers to arecombinant molecule that comprises an antibody component and a zB7H6polypeptide component. Examples of an antibody fusion protein include aprotein that comprises a zB7H6 extracellular domain, and either an Fcdomain or an antigen-binding region.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

An “inhibitory polynucleotide” is a DNA or RNA molecule that reduces orprevents expression (transcription or translation) of a second (target)polynucleotide. Inhibitory polynucleotides include antisensepolynucleotides, ribozymes, and external guide sequences. The term“inhibitory polynucleotide” further includes DNA and RNA molecules thatencode the actual inhibitory species, such as DNA molecules that encoderibozymes.

An “anti-sense oligonucleotide specific for zB7H6” or a “zB7H6anti-sense oligonucleotide” is an oligonucleotide having a sequence (a)capable of forming a stable triplex with a portion of the zB7H6 gene, or(b) capable of forming a stable duplex with a portion of an mRNAtranscript of the zB7H6 gene.

A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

An “external guide sequence” is a nucleic acid molecule that directs theendogenous ribozyme, RNase P, to a particular species of intracellularmRNA, resulting in the cleavage of the mRNA by RNase P. A nucleic acidmolecule that encodes an external guide sequence is termed an “externalguide sequence gene.”

The term “variant zB7H6 gene” refers to nucleic acid molecules thatencode a polypeptide having an amino acid sequence that is amodification of SEQ ID NO:2. Such variants include naturally-occurringpolymorphisms of zB7H6 genes, as well as synthetic genes that containconservative amino acid substitutions of the amino acid sequence of SEQID NO:2. Additional variant forms of zB7H6 genes are nucleic acidmolecules that contain insertions or deletions of the nucleotidesequences described herein. A variant zB7H6 gene can be identified, forexample, by determining whether the gene hybridizes with a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, or itscomplement, under stringent conditions.

Alternatively, variant zB7H6 genes can be identified by sequencecomparison. Two amino acid sequences have “100% amino acid sequenceidentity” if the amino acid residues of the two amino acid sequences arethe same when aligned for maximal correspondence. Similarly, twonucleotide sequences have “100% nucleotide sequence identity” if thenucleotide residues of the two nucleotide sequences are the same whenaligned for maximal correspondence. Sequence comparisons can beperformed using standard software programs such as those included in theLASERGENE bioinformatics computing suite, which is produced by DNASTAR(Madison, Wis.). Other methods for comparing two nucleotide or aminoacid sequences by determining optimal alignment are well-known to thoseof skill in the art. (See, e.g., Peruski and Peruski, The Internet andthe New Biology: Tools for Genomic and Molecular Research (ASM Press,Inc. 1997); Wu et al. (eds.), “Information Superhighway and ComputerDatabases of Nucleic Acids and Proteins,” in Methods in GeneBiotechnology 123-151 (CRC Press, Inc. 1997); Bishop (ed.), Guide toHuman Genome Computing (2nd ed., Academic Press, Inc. 1998).) Twonucleotide or amino acid sequences are considered to have “substantiallysimilar sequence identity” or “substantial sequence identity” if the twosequences have at least 80%, at least 90%, or at least 95% sequenceidentity relative to each other. Particular methods for determiningsequence identity are described below.

Regardless of the particular method used to identify a variant zB7H6gene or variant zB7H6 polypeptide, a variant gene or polypeptide encodedby a variant gene may be functionally characterized the ability to bindspecifically to an anti-zB7H6 antibody. A variant zB7H6 gene or variantzB7H6 polypeptide may also be functionally characterized by the abilityto bind to NKp30, using a biological or biochemical assay such asdescribed herein.

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The term “ortholog” denotes a polypeptide or protein obtained from onespecies that is the functional counterpart of a polypeptide or proteinfrom a different species. Sequence differences among orthologs are theresult of speciation.

“Paralogs” are distinct but structurally related proteins made by anorganism. Paralogs are believed to arise through gene duplication. Forexample, α-globin, β-globin, and myoglobin are paralogs of each other.

“NK cell activity” as used herein refers to NK cell cytolytic activity.There are numerous assays well-known to the skilled artisan fordetecting and/or monitoring such activity, including but not limited tothe assays described in the examples provided herein.

As used herein, the phrase “interaction of zB7H6 and NKp30” refers todirect physical interaction (e.g., binding) and/or other indirectinteraction of a functional zB7H6 receptor with NKp30 on an NK cell,resulting in stimulation of the zB7H6 receptor and/or NKp30 andassociated intracellular signaling.

As used herein, the term “blocking agent” includes those agents thatinterfere with the interaction of zB7H6 and NKp30, and/or that interferewith the ability of the zB7H6 to trigger NK cell activity, e.g., asmeasured by cytolytic activity. Exemplary agents includefunction-blocking antibodies, as well as peptides that block the bindingzB7H6 with NKp30 but that fail to stimulate zB7H6-mediated signaling inan NK cell (e.g., zB7H6-derived peptides, peptidomimetics, smallmolecules, and the like).

As used herein, the term “mimicking agent” includes those agents thatmimic the interaction of zB7H6 and NKp30, and/or augment, enhance orincrease the ability of zB7H6 and/or NKp30 to trigger NK cell activity.Exemplary agents include zB7H6 soluble receptors, peptides that augmentor enhance the ability of zB7H6 to bind to NKp30 or substitute for zB7H6in stimulating NKp30-mediated signaling (e.g., B7H6-derived peptides,peptidomimetics, small molecules, and the like), and zB7H6anti-idiotypic antibodies.

The present invention includes functional fragments of zB7H6polypeptides. Within the context of this invention, a “functionalfragment” of a zB7H6 refers to a portion of a zB7H6 polypeptide that atleast specifically binds to NKp30. In some embodiments, a functionalfragment of zB7H6 is capable of triggering or enhancing NKp30-mediatedNK cell activation; in other embodiments, a functional fragment iscapable of blocking or decreasing NKp30-mediated NK cell activation.

The term “zB7H6-related agent” or “zB7H6-related composition,” as usedherein, refers to an agent that demonstrates zB7H6 functional activityor inhibition of zB7H6 functional activity, or an agent thatdemonstrates zB7H6-specific binding. Such agents include, for example,soluble zB7H6 polypeptides, anti-zB7H6 antibodies, anti-zB7H6antibody-drug conjugates, zB7H6 anti-idiotypic antibodies or other zB7H6mimicking agents, zB7H6-encoding polynucleotides, inhibitorypolynucleotides, and the like.

The phrase “demonstrates zB7H6 functional activity” or “demonstrateszB7H6 activity,” in reference to an agent or composition, refersgenerally to zB7H6 mimicking agents (including, e.g., soluble zB7H6polypeptides and zB7H6 anti-idiotypic antibodies) as well aspolynucleotides encoding polypeptides that have zB7H6 functionalactivity.

The phrase “demonstrates inhibition of zB7H6 functional activity,” inreference to an agent or composition, refers generally to zB7H6 blockingagents (including, e.g., function-blocking anti-zB7H6 antibodies andpeptides that block the binding zB7H6 with NKp30 but that fail tostimulate zB7H6-mediated signaling) as well as nucleic acids that reduceor prevent expression of a zB7H6 gene (i.e., zB7H6 inhibitorypolynucleotides).

The term “NK cell-associated disease or disorder,” as used herein,refers to generally to NK-cell-mediated diseases or disorders as well asdiseases or disorders characterized by insufficient NK cell activity.

The phrase “disease or disorder characterized by insufficient NK cellactivity,” as used herein, refers to any disease or disorder thatinvolves, at least in part, pathogenic cells that can serve as targetsfor NK cell cytolytic activity, but which are prominent in the diseaseor disorder at least partly as a result of having evaded NKcell-mediated cytotoxicity. Such pathogenic cells are typically thoselacking MHC class I expression, such as, for example, certain tumorcells or virus-infected cells. Accordingly, typical diseases ordisorders characterized by insufficient NK cell activity are cancers andmany infectious diseases. Such diseases and disorders are particularlyamenable to certain treatment methods for enhancing NK cell activity, asdescribed further herein.

The term “NK cell-mediated disease or disorder,” as used herein, refersto any disease or disorder having a pathology that is mediated, at leastin part, by NK cell cytolytic activity. An example of such a disease ordisorder is acute rejection of bone marrow cell (BMC) allografts. Suchdiseases or disorder are particularly amenable to certain treatmentmethods for inhibition NK cell activity, as described further herein.

The term “effective amount,” in the context of treatment of a NKcell-associated disease or disorder by administration of a soluble zB7H6polypeptide or an antibody to a subject as described herein, refers toan amount of such molecule that is sufficient to modulate an NKcell-mediated response in the subject so as to inhibit the occurrence orameliorate one or more symptoms of the NK cell-associated disease ordisorder. An effective amount of an agent is administered according tothe methods of the present invention in an “effective regime.” The term“effective regime” refers to a combination of amount of the agent beingadministered and dosage frequency adequate to accomplish treatment orprevention of the disease or disorder.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to ±10%.

III. zB7H6 Polypeptides, Nucleic Acids, Vectors, Host Cells, and RelatedMethods for Production

The zB7H6 polypeptides of the present invention generally comprise thezB7H6 extracellular domain (residues 25-266 of SEQ ID NO:2), or afunctional variant or fragment thereof. Such zB7H6 polypeptides areuseful, for example, in the modulation of NK cell activity and in thetreatment of disorders such as cancer or infectious disease, as well asin methods of screening agents for activity against the functionalinteraction of zB7H6 with NKp30. Generally, zB7H6 polypeptides of theinvention comprise a polypeptide region selected from the following:

-   -   (i) the extracellular domain of the zB7H6 polypeptide of SEQ ID        NO:2 (i.e., residues 25-266 of SEQ ID NO:2);    -   (ii) a functional variant of the zB7H6 extracellular domain of        (i), the variant having at least 80% identity with residues        25-266 of SEQ ID NO:2; and    -   (iii) a functional fragment of the zB7H6 extracellular domain        of (i) or of the domain variant of (ii).

In certain embodiments, a zB7H6 polypeptide is a soluble receptorpolypeptide. Such soluble forms of zB7H6 lack a functional transmembranedomain and typically are also substantially free of intracellularpolypeptide segments. In some alternative embodiments, a zB7H6polypeptide is a cell membrane-bound form of zB7H6, such as, e.g., azB7H6 polypeptide comprising a functional transmembrane domain or a GPIlinkage. Cell-membrane bound forms of zB7H6 include, for example, fulllength and substantially full-length forms of zB7H6 protein, such as apolypeptide comprising or consisting of residues 25-454 of SEQ ID NO:2,or a variant thereof.

In some embodiments of a zB7H6 polypeptide comprising a functionalextracellular domain variant, the variant has at least 85%, at least90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity with residues 25-266 of SEQ ID NO:2. Similarly, inother embodiments comprising a functional fragment of an extracellulardomain variant, the fragment is derived from a variant having at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity with residues 25-266 of SEQ IDNO:2. As previously indicated, in certain embodiments, a zB7H6polypeptide can further comprise transmembrane and intracellular domaincomponents; in some such embodiments, a polypeptide of the invention hasat least 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity with residues 25-454 of SEQID NO:2.

Percent sequence identity is determined by conventional methods. See,e.g., Altschul et al., Bull. Math. Bio. 48:603, 1986, and Henikoff andHenikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1992. For example, twoamino acid sequences can be aligned to optimize the alignment scoresusing a gap opening penalty of 10, a gap extension penalty of 1, and the“BLOSUM62” scoring matrix of Henikoff and Henikoff, supra, as shown inTable 1 (amino acids are indicated by the standard one-letter codes).The percent identity is then calculated as: ([Total number of identicalmatches]/[length of the longer sequence plus the number of gapsintroduced into the longer sequence in order to align the twosequences]) (100).

TABLE 1 BLOSUM62 Scoring Matrix A R N D C Q E G H I L K M F P S T W Y VA 4 R −1 5 N −2 0 6 D −2 −2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 02 −4 2 5 G 0 −2 0 −1 −3 −2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1−3 −3 −4 −3 4 L −1 −2 −3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1−3 −2 5 M −1 −1 −2 −3 −1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3−1 0 0 −3 0 6 P −1 −2 −2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0−1 0 0 0 −1 −2 −2 0 −1 −2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2−1 1 5 W −3 −3 −4 −4 −2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2−3 −2 −1 −2 −3 2 −1 −1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −33 1 −2 1 −1 −2 −2 0 −3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativezB7H6 variant. The FASTA algorithm is described by Pearson and Lipman,Proc. Nat'l Acad. Sci. USA 85:2444, 1988, and by Pearson, Meth. Enzymol.183:63, 1990. Briefly, FASTA first characterizes sequence similarity byidentifying regions shared by the query sequence (e.g., residues 25-266of SEQ ID NO:2) and a test sequence that have either the highest densityof identities (if the ktup variable is 1) or pairs of identities (ifktup=2), without considering conservative amino acid substitutions,insertions, or deletions. The ten regions with the highest density ofidentities are then rescored by comparing the similarity of all pairedamino acids using an amino acid substitution matrix, and the ends of theregions are “trimmed” to include only those residues that contribute tothe highest score. If there are several regions with scores greater thanthe “cutoff” value (calculated by a predetermined formula based upon thelength of the sequence and the ktup value), then the trimmed initialregions are examined to determine whether the regions can be joined toform an approximate alignment with gaps. Finally, the highest scoringregions of the two amino acid sequences are aligned using a modificationof the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol.Biol. 48:444, 1970; Sellers, SIAM J. Appl. Math. 26:787, 1974), whichallows for amino acid insertions and deletions. Illustrative parametersfor FASTA analysis are: ktup=1, gap opening penalty=10, gap extensionpenalty=1, and substitution matrix=BLOSUM62. These parameters can beintroduced into a FASTA program by modifying the scoring matrix file(“SMATRIX”), as explained in Appendix 2 of Pearson, Meth. Enzymol.183:63, 1990.

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdescribed above.

The present invention includes soluble zB7H6 polypeptides having aconservative amino acid change compared with the amino acid sequence ofSEQ ID NO:2 residues 25-266. For example, zB7H6 variants can be obtainedthat contain one or more amino acid substitutions of SEQ ID NO:2residues 25-266 in which an alkyl amino acid is substituted for an alkylamino acid in a zB7H6 amino acid sequence, an aromatic amino acid issubstituted for an aromatic amino acid in a zB7H6 amino acid sequence, asulfur-containing amino acid is substituted for a sulfur-containingamino acid in a zB7H6 amino acid sequence, a hydroxy-containing aminoacid is substituted for a hydroxy-containing amino acid in a zB7H6 aminoacid sequence, an acidic amino acid is substituted for an acidic aminoacid in a zB7H6 amino acid sequence, a basic amino acid is substitutedfor a basic amino acid in a zB7H6 amino acid sequence, or a dibasicmonocarboxylic amino acid is substituted for a dibasic monocarboxylicamino acid in a zB7H6 amino acid sequence. Among the common amino acids,a “conservative amino acid substitution” is illustrated by, for example,a substitution among amino acids within each of the following groups:

(1) glycine, alanine, valine, leucine, and isoleucine, (2)phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4)aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine,arginine and histidine. Exemplary groups of conservative amino acidchanges are further shown in Table 2 below.

TABLE 2 Conservative amino acid substitutions Basic Acidic PolarHydrophobic Aromatic Small arginine glutamate glutamine leucinephenylalanine glycine lysine aspartate asparagine isoleucine tryptophanalanine histidine valine tyrosine serine methionine threioninemethionine

The BLOSUM62 table is an amino acid substitution matrix derived fromabout 2,000 local multiple alignments of protein sequence segments,representing highly conserved regions of more than 500 groups of relatedproteins (Henikoff and Henikoff, Proc. Nat'l Acad. Sci. USA 89:10915,1992). Accordingly, the BLOSUM62 substitution frequencies can be used todefine conservative amino acid substitutions that may be introduced intothe amino acid sequences of the present invention. Although it ispossible to design amino acid substitutions based solely upon chemicalproperties (as discussed above), the language “conservative amino acidsubstitution” preferably refers to a substitution represented by aBLOSUM62 value of greater than −1. For example, an amino acidsubstitution is conservative if the substitution is characterized by aBLOSUM62 value of 0, 1, 2, or 3. According to this system, preferredconservative amino acid substitutions are characterized by a BLOSUM62value of at least 1 (e.g., 1, 2 or 3), while more preferred conservativeamino acid substitutions are characterized by a BLOSUM62 value of atleast 2 (e.g., 2 or 3). Particular variants of zB7H6 are characterizedby having at least 80%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to thecorresponding amino acid sequence (e.g., residues 25-266 of SEQ IDNO:2), wherein the variation in amino acid sequence is due to one ormore conservative amino acid substitutions.

A functional zB7H6 variant or fragment polypeptide can be readilyidentified using routine assays for assessing the ability of the variantor fragment to specifically bind to NKp30 (e.g., human NKp30), and/orassays to assess the ability of the variant or fragment to triggerNKp30-mediated NK cell activation. For example, cells expressing NKp30can be probed by FACS using soluble zB7H6 polypeptide, which may bedirectly labeled or detected using a secondary reagent specific for amoiety of the soluble zB7H6 polypeptide (e.g., a fluorophore-conjugatedstreptavidin to detect biotinylated zB7H6 polypeptide, or afluorophore-conjugated anti-IgG antibody to detect a zB7H6 fusionprotein comprising an Fc fragment). In other variations, functionalzB7H6 polypeptides may be identified by their ability to trigger NK cellcytolytic activity against target cells. Exemplary assays for assessingzB7H6-related function of zB7H6 variants and fragments are furtherdescribed herein.

In certain variations, a soluble zB7H6 polypeptide is a fusion proteincomprising the zB7H6 extracellular domain, or the functional variant orfragment thereof, and a heterologous polypeptide. Suitable heterologouspolypeptides include immunoglobulin heavy chain constant regions. Forexample, in some embodiments the immunoglobulin heavy chain constantregion is an F_(c) fragment (e.g., a human F_(c) fragment), whichcontains two or three constant region domains and a hinge region butlacks the variable region. (See, e.g., U.S. Pat. Nos. 6,018,026 and5,750,375 to Sledziewski et al.) Such fusions comprising F_(c) fragmentsare typically secreted as multimeric, typically dimeric, moleculeswherein the F_(c) portions are disulfide bonded to each other and tworeceptor polypeptides are arrayed in closed proximity to each other. Asan illustration, U.S. Pat. No. 5,723,125 (Chang et al.) describes afusion protein comprising a human interferon and a human immunoglobulinF_(c) fragment. The C-terminus of the interferon is linked to theN-terminus of the F_(c) fragment by a peptide linker moiety. An exampleof a peptide linker is a peptide comprising primarily a T cell inertsequence, which is immunologically inert. An illustrative F_(c) moietyis a human γ4 chain, which is stable in solution and has little or nocomplement activating activity. Other suitable F_(c) moieties includevariants of the human γ1 chain that lack or have substantially reducedeffector function, such as, for example, Fc4 (SEQ ID NO:31), Fc5 (SEQ IDNO:32), Fc6 (SEQ ID NO:33), and Fc7 (SEQ ID NO:34), which are depictedin FIGS. 13A-13C. Accordingly, in some embodiments, the presentinvention provides a zB7H6 fusion protein that comprises the zB7H6extracellular domain, or the functional variant or fragment thereof, andan F_(c) fragment (e.g., a human F_(c) fragment or variant thereof),wherein the C-terminus of the zB7H6 extracellular domain, or thefunctional variant or fragment thereof, is attached to the N-terminus ofthe F_(c) fragment via a peptide linker.

Other particularly suitable heterologous polypeptides for production ofsoluble B7H6 fusion proteins include VASP domains. The use of VASPdomains in soluble receptor fusion proteins is described in more detailin U.S. Patent Application Publication No. 2007/0254339, which isincorporated by reference herein in its entirety. VASP domains arederived from the VASP gene present in many species. Sequences areselected for their anticipated ability to form coiled-coil proteinstructure, as this structure is important for the ability to formmultimeric protein forms. Particularly desired for the present inventionis the ability of coiled-coil proteins to produce tetrameric proteinstructures. A particularly preferred embodiment utilizes amino acids 342to 375 of the human VASP sequence, the full-length polypeptide sequenceof which is set forth in SEQ ID NO:4. The full length DNA sequenceencoding the human VASP protein is set forth in SEQ ID NO:3.

Work with other types of multimerizing sequences, for examples, theleucine zipper, has shown that a limited number of conservative aminoacid substitutions (even at the d residue) can be often be tolerated inzipper sequences without the loss of the ability of the molecules tomultimerize (Landschulz et al., Science 243:1681-1688, 1989). Thus,conservative changes from the native sequence for the VASP domain arecontemplated within the scope of the invention. For example, Table 2,supra, shows exemplary conservative changes that are predicted to betolerated by the coiled-coil structure.

If more than one fusion protein is being used to produce ahetero-multimeric protein, for example, heterotetramers, the VASP domainthat is used can be the same domain for both fusion proteins ordifferent VASP domains, as long as the domains have the ability toassociate with each other and form multimeric proteins.

In certain embodiments, the VASP domain is linked at the C terminus ofthe zB7H6 extracellular domain as shown in residues 25-266 of SEQ IDNO:2 (or to a functional variant or fragment thereof). Additionally, theVASP domain can be located in the middle of the protein, effectivelycreating a double fusion protein with a VASP domain flanked by twonon-VASP polypeptide segments, where at least one of the polypeptidesegments flanking the VASP domain is the zB7H6 extracellular domain asshown in residues 25-266 of SEQ ID NO:2 (or to a functional variant orfragment thereof). In some variations, the second polypeptide segmentflanking the VASP domain is a polypeptide segment designed to target thesoluble receptor to specific cells or tissues for the benefit of zB7H6binding activity.

One result of the use of multimerizing heterologous polypeptidesequences in soluble zB7H6 fusion constructs is the ability to increasethe affinity or avidity of zB7H6 for a ligand or counter-receptor (e.g.,NKp30) through the formation of a multimeric form. By avidity, it ismeant the strength of binding of multiple molecules to a largermolecule, a situation exemplified but not limited to the binding of acomplex antigen by an antibody. By affinity, it is meant the strength ofbinding of a simple receptor-ligand system. Such a characteristic wouldbe improved, for example, by forming a binding site with better bindingcharacteristics for zB7H6 through multimerization of the receptor.Avidity and affinity can be measured using standard assays well known toone of ordinary skill. An improvement in affinity or avidity occurs whenthe affinity or avidity value (for example, affinity constant or K_(a))for the multimeric soluble zB7H6 fusion protein and a ligand orcounter-receptor is higher than for a monomeric zB7H6 polypeptide andthe ligand or counter-receptor. An alternative means of measuring thesecharacteristics is the equilibrium constant (K_(d)) where a decreasewould be observed with the improvement in affinity or avidity using amultimerizing heterologous polypeptide (e.g., a VASP tetramerizationdomain).

Polypeptide segments of a soluble zB7H6 fusion protein (e.g., a zB7H6extracellular domain, or functional variant or fragment thereof, and asegment heterologous to zB7H6) may be linked directly to another proteinto form the fusion protein; alternatively, the polypeptide segments maybe separated by a distance sufficient to ensure that the proteins formproper secondary and tertiary structure needed for biological activity.Suitable linker sequences will adopt a flexible extended confirmationand will not exhibit a propensity for developing an ordered secondarystructure which could interact with the functional domains of thefusions proteins, and will have minimal hydrophobic or charged characterwhich could also interfere with the function of fusion domains. Linkersequences should be constructed with the 15 residue repeat in mind, asit may not be in the best interest of producing a biologically activeprotein to tightly constrict the N or C terminus of the heterologoussequence. Beyond these considerations, the length of the linker sequencemay vary without significantly affecting the biological activity of thefusion protein. Linker sequences can be used between any and allcomponents of the fusion protein (or expression construct) includingaffinity tags and signal peptides. An example linker is the GSGGsequence (SEQ ID NO:5).

A soluble zB7H6 fusion protein can further include an affinity tag. Suchtags do not alter the biological activity of fusion proteins, are highlyantigenic, and provide an epitope that can be reversibly bound by aspecific binding molecule, such as a monoclonal antibody, facilitatingrapid detection and purification of an expressed fusion protein.Affinity tags can also convey resistance to intracellular degradation ifproteins are produced in bacteria, such as E. coli. An exemplaryaffinity tag is the FLAG Tag (SEQ ID NO:6) or the HIS₆ Tag (SEQ IDNO:7). Methods of producing fusion proteins utilizing this affinity tagfor purification are described in U.S. Pat. No. 5,011,912.

In some variations, a soluble zB7H6 receptor comprises a “targetingdomain,” a heterologous polypeptide segment designed to target thesoluble receptor to specific cells or tissues for the benefit of zB7H6binding activity. For example, in some embodiments, the soluble fusionprotein comprises a polypeptide segment that specifically targets thefusion protein to tumor cells. Particularly suitable heterologouspolypeptide segments for targeting fusion proteins to particular cellsor tissues include antibodies or antigen-binding fragments thereof thatrecognize cell surface markers associated with the target cells ortissues. The use of targeting domains can provide a high localconcentration of a soluble zB7H6 receptor in the vicinity of a targettissue (e.g., a tumor), thereby reducing the amount of soluble receptorthat must be administered to effect a desired response as well asminimizing undesired side effects that may be caused by exposure ofnon-target tissues to the soluble receptor. In addition, the binding ofa targeting domain portion of a zB7H6 fusion protein to the surface of atarget cell may enhance cross-linking of zB7H6-bound NKp30 on thesurface of NK cells, thereby further enhancing NKp30-mediatedstimulation of NK cell activity against the target cell.

For example, in the case of a tumor target tissue, targeting domains caninclude tumor-specific or tumor-associated antigens (i.e., antigens thatare expressed by tumor cells but not normal cells, or antigens that areexpressed at high levels in tumor cells relative to normal cells).Examples of such antigens include epidermal growth factor receptorfamily members (e.g., EGFR and Her2), carcinoembryonic antigen (CEA),members of the mucin family (MUC1), mesothelin, follate receptor, andothers. Antigens that are specific to or associated with hematopoietictumors could also be targeted, including, for example, CD30, CD33, CD40,CD72, and others. Antibodies against all these antigens are eitherapproved or in clinical trials for the treatment of multiple cancers. AzB7H6 fusion protein comprising an antibody against at least one ofthese surface receptors would enable local targeting of the molecule,and could further facilitate cross-linking of zB7H6-bound NKp30 on thesurface of NK cells, thereby further enhancing NKp30-mediatedstimulation of NK cell activity against tumor cells.

The present invention further provides a variety of other polypeptidefusions. For example, in some embodiments, a zB7H6 polypeptide can befused to two or more moieties or domains, such as an affinity tag forpurification and a targeting domain. Polypeptide fusions can alsocomprise one or more cleavage sites, particularly between domains. See,e.g., Tuan et al., Connective Tissue Research 34:1 (1996).

In some variations, a zB7H6 polypeptide further comprises a signalsequence or leader sequence. These sequences are generally utilized toallow for secretion of the fusion protein from the host cell duringexpression and are also known as a leader sequence, prepro sequence orpre sequence. While the secretory signal sequence may be derived fromzB7H6, a suitable signal sequence may also be derived from anothersecreted protein (e.g., the tissue-type plasminogen activator (t-PA)signal sequence, as described, for example, in U.S. Pat. No. 5,641,655)or synthesized de novo. The secretory signal sequence is operably linkedto a zB7H6-encoding sequence such that the two sequences are joined inthe correct reading frame and positioned to direct the newly synthesizedpolypeptide into the secretory pathway of the host cell. Secretorysignal sequences are commonly positioned 5′ to the nucleotide sequenceencoding the polypeptide of interest, although certain secretory signalsequences may be positioned elsewhere in the nucleotide sequence ofinterest (see, e.g., U.S. Pat. No. 5,037,743 to Welch et al.; U.S. Pat.No. 5,143,830 to Holland et al.)

Although the secretory signal sequence of zB7H6 or another proteinproduced by mammalian cells (e.g., tissue-type plasminogen activatorsignal sequence, as described, for example, in U.S. Pat. No. 5,641,655)is useful for expression of zB7H6 in recombinant mammalian hosts, ayeast signal sequence is preferred for expression in yeast cells.Examples of suitable yeast signal sequences are those derived from yeastmating phermone α-factor (encoded by the MFα1 gene), invertase (encodedby the SUC2 gene), or acid phosphatase (encoded by the PHO5 gene). See,e.g., Romanos et al., “Expression of Cloned Genes in Yeast,” in DNACloning 2: A Practical Approach, 2^(nd) Edition, Glover and Hames(eds.), pages 123-167 (Oxford University Press 1995).

In some variations, zB7H6 polypeptides are chemically modified vialinkage to a polymer. Typically, the polymer is water soluble so thatthe zB7H6 polypeptide conjugate does not precipitate in an aqueousenvironment, such as a physiological environment. An example of asuitable polymer is one that has been modified to have a single reactivegroup, such as an active ester for acylation, or an aldehyde foralkylation. In this way, the degree of polymerization can be controlled.The polymer may be branched or unbranched. A zB7H6 polypeptide conjugatecan also comprise a mixture of such water-soluble polymers. Generalmethods for producing conjugates comprising a polypeptide andwater-soluble polymer moieties are known in the art. (See, e.g., U.S.Pat. No. 5,382,657 to Karasiewicz et al.; U.S. Pat. No. 5,738,846 toGreenwald et al.; Nieforth et al., Clin. Pharmacol. Ther. 59:636, 1996;Monkarsh et al., Anal. Biochem. 247:434, 1997.) Such methods can beemployed for making zB7H6-comprising homodimeric, heterodimeric ormultimeric soluble receptor conjugates.

One example of a zB7H6 polypeptide conjugate comprises a polyalkyl oxidemoiety attached to the N-terminus of the zB7H6 polypeptide. PEG is onesuitable polyalkyl oxide. As an illustration, zB7H6 can be modified withPEG, a process known as “PEGylation.” PEGylation of zB7H6 can be carriedout by any of the PEGylation reactions known in the art. (See, e.g., EP0 154 316; Delgado et al., Critical Reviews in Therapeutic Drug CarrierSystems 9:249, 1992; Duncan and Spreafico, Clin. Pharmacokinet. 27:290,1994; Francis et al., Int J Hematol 68:1, 1998.) For example, PEGylationcan be performed by an acylation reaction or by an alkylation reactionwith a reactive polyethylene glycol molecule. In an alternativeapproach, zB7H6 conjugates are formed by condensing activated PEG, inwhich a terminal hydroxy or amino group of PEG has been replaced by anactivated linker. (See, e.g., U.S. Pat. No. 5,382,657 to Karasiewicz etal.) For PEGylation reactions, the typical molecular weight of a polymermolecule is about 2 kDa to about 100 kDa, about 5 kDa to about 50 kDa,or about 12 kDa to about 25 kDa. The molar ratio of water-solublepolymer to zB7H6 will generally be in the range of 1:1 to 100:1.Typically, the molar ratio of water-soluble polymer to zB7H6 will be 1:1to 20:1 for polyPEGylation, and 1:1 to 5:1 for monoPEGylation.

zB7H6 polypeptides can be used, for example, to affinity purify acognate counter-receptor (e.g., NKp30) from solution, or as an in vitroassay tool. For example, the presence of a zB7H6 counter-receptor in abiological sample can be detected using a zB7H6-immunoglobulin fusionprotein, in which the zB7H6 moiety is used to bind the counter-receptor,and a macromolecule, such as Protein A or anti-Fc antibody, is used tobind the fusion protein to a solid support. Such systems can be used toidentify agonists and antagonists that interfere with the binding ofzB7H6 to its counter-receptor (e.g., NKp30).

zB7H6 polypeptides can also be used to trigger or enhance signals invitro by specifically binding NKp30 on cells, and as agonists in vivo byadministering them parenterally (e.g., by intramuscular, subcutaneous orintravenous injection) to bind NKp30 on cells and trigger or enhanceNKp30-mediated activation of NK cells. For example, a soluble zB7H6fusion protein can be used for triggering or enhancing NK cell cytolyticactivity in vitro, or for triggering or enhancing such activity ex vivoor in vivo for treatment of cancer or infectious disease. These andother uses are described further herein.

Using methods as discussed herein, one of ordinary skill in the art canprepare a variety of zB7H6 polypeptides as described herein, includingpolypeptides that comprise the zB7H6 extracellular domain of SEQ ID NO:2residues 25-266, or a zB7H6 extracellular domain substantially identicalthereto and retaining the NKp30-binding or other functional propertiesof SEQ ID NO:2 residues 25-266. The zB7H6 polypeptides of the inventionare typically recombinantly produced, although such polypeptides canalso be produced by other methods generally available in the art (e.g.,synthetic production of polypeptides, or by isolation of zB7H6polypeptides from natural sources). Recombinant zB7H6 receptorpolypeptides can generally be prepared by expressing a polynucleotidecomprising a DNA segment encoding the zB7H6 polypeptide. For example,recombinant zB7H6 soluble receptor polypeptides can generally beprepared by expressing a polynucleotide comprising a truncated DNAencoding the extracellular domain of the zB7H6 polypeptide of SEQ IDNO:2 (contiguous amino acid residues 25-266 of SEQ ID NO:2), or afunctional variant or fragment thereof. As it is preferred that thesoluble extracellular domain polypeptides be prepared in a formsubstantially free of transmembrane and intracellular polypeptidesegments, polynucleotides encoding such a soluble polypeptide willtypically lack regions encoding such transmembrane and intracellularsegments. Methods for recombinant production of protein are generallywell-known in the art.

As discussed above, soluble zB7H6 polypeptides may also includeadditional polypeptide segments as generally disclosed herein. In thecase of soluble zB7H6 fusion proteins, such embodiments can also beprepared by methods generally known to those skilled in the art. Forexample, fusion proteins can be prepared by preparing each component ofthe fusion protein and chemically conjugating them. General methods forenzymatic and chemical cleavage of fusion proteins are described, forexample, by Ausubel (1995) at pages 16-19 to 16-25. Alternatively, apolynucleotide encoding both components of the fusion protein in theproper reading frame can be generated using known techniques andrecombinantly expressed using methods such as further described furtherherein.

As indicated above, zB7H6 receptor polypeptides can generally beprepared by expressing a polynucleotide comprising a DNA segmentencoding the zB7H6 polypeptide. For soluble protein forms, it ispreferred that the extracellular domain polypeptide be prepared in aform substantially free of transmembrane and intracellular polypeptidesegments. To direct the export of the receptor domain from the hostcell, the receptor DNA is linked to a second DNA segment encoding asecretory peptide, such as a t-PA secretory peptide. In someembodiments, to facilitate purification of the secreted receptor domain,a C-terminal extension, such as a poly-histidine tag, substance P, FLAG™peptide (Hopp et al., Biotechnology 6:1204-1210, 1988; available fromEastman Kodak Co., New Haven, Conn.) or another polypeptide or proteinfor which an antibody or other specific binding agent is available, canbe fused to the receptor polypeptide.

Accordingly, in another aspect, the present invention further providespolynucleotides encoding any of the zB7H6 polypeptides as describedherein. Generally, polynucleotides encoding a soluble zB7H6 polypeptidecomprises a polynucleotide region encoding the extracellular zB7H6domain of residues 25-266 of SEQ ID NO:2, or a functional variant orfragment thereof. In certain other variations, a polynucleotide of theinvention encodes a cell-membrane bound form of zB7H6, such as apolypeptide comprising residues 25-454 or 1-454 of SEQ ID NO:2, or afunctional variant thereof. In a specific embodiment, a polynucleotideencoding a soluble zB7H6 polypeptide comprises nucleotide residues73-798 or 1-798 of SEQ ID NO:1; examples of polynucleotides encodingresidues 25-454 or 1-454 of SEQ ID NO:2 include polynucleotidescomprising 73-1362 or 1-1362 of SEQ ID NO:1. In certain variations,polynucleotides of the invention further include one or morepolynucleotide regions encoding additional component(s) of a zB7H6polypeptide, such as, for example, a heterologous polypeptide componentof a zB7H6 fusion protein, a signal secretory sequence, and/or anaffinity tag.

As will be appreciated by those in the art, due to the degeneracy of thegenetic code, an extremely large number of nucleic acids may be made,all of which encode the zB7H6 polypeptides of the present invention.Thus, given a particular amino acid sequence of a zB7H6 polypeptide, anynumber of different nucleic acids encoding the polypeptide can be madeusing known techniques to modify the sequence of one or more codons in away which does not change the amino acid sequence of a zB7H6polypeptide.

A zB7H6-encoding cDNA can be isolated by a variety of methods, such asby probing with a complete or partial human cDNA or with one or moresets of degenerate probes based on the disclosed sequences. A cDNA canalso be cloned using the polymerase chain reaction with primers designedfrom the representative human zB7H6 sequences disclosed herein. Inaddition, a cDNA library can be used to transform or transfect hostcells, and expression of the cDNA of interest can be detected with anantibody to zB7H6 polypeptide.

For example, nucleic acid molecules encoding a human zB7H6 gene can beobtained by screening a human cDNA or genomic library usingpolynucleotide probes based upon SEQ ID NO:1. These techniques arestandard and well-established, and may be accomplished using cloningkits available by commercial suppliers. See, e.g., Ausubel et al.(eds.), Short Protocols in Molecular Biology (3^(rd) ed., John Wiley &Sons 1995); Wu et al., Methods in Gene Biotechnology, CRC Press, Inc.1997; Aviv and Leder, Proc. Nat'l Acad. Sci. USA 69:1408, 1972; Huynh etal., “Constructing and Screening cDNA Libraries in λgt10 and λgt11,” inDNA Cloning: A Practical Approach Vol. I, Glover (ed.), page 49 (IRLPress, 1985).

Nucleic acid molecules that encode a human zB7H6 gene can also beobtained using the polymerase chain reaction (PCR) with oligonucleotideprimers having nucleotide sequences that are based upon the nucleotidesequences of the zB7H6 gene or cDNA. General methods for screeninglibraries with PCR are provided by, for example, Yu et al., “Use of thePolymerase Chain Reaction to Screen Phage Libraries,” in Methods inMolecular Biology, Vol. 15: PCR Protocols: Current Methods andApplications (White, ed., Humana Press, Inc. 1993). Moreover, techniquesfor using PCR to isolate related genes are described by, for example,Preston, “Use of Degenerate Oligonucleotide Primers and the PolymeraseChain Reaction to Clone Gene Family Members,” in Methods in MolecularBiology, Vol. 15: PCR Protocols: Current Methods and Applications(White, ed., Humana Press, Inc. 1993). As an alternative, a zB7H6 genecan be obtained by synthesizing nucleic acid molecules using mutuallypriming long oligonucleotides and the nucleotide sequences describedherein (see, e.g., Ausubel, supra). Established techniques using thepolymerase chain reaction provide the ability to synthesize DNAmolecules at least two kilobases in length. (See, e.g., Adang et al.,Plant Molec. Biol. 21:1131, 1993; Bambot et al., PCR Methods andApplications 2:266, 1993; Dillon et al., “Use of the Polymerase ChainReaction for the Rapid Construction of Synthetic Genes,” in Methods inMolecular Biology, Vol. 15: PCR Protocols: Current Methods andApplications 263-268 (White, ed., Humana Press, Inc. 1993); andHolowachuk et al., PCR Methods Appl. 4:299, 1995.) For reviews onpolynucleotide synthesis, see, for example, Glick and Pasternak,Molecular Biotechnology, Principles and Applications of Recombinant DNA(ASM Press 1994); Itakura et al., Annu. Rev. Biochem. 53:323, 1984; andClimie et al., Proc. Nat'l Acad. Sci. USA 87:633, 1990.

As previously discussed, those skilled in the art will readily recognizethat, in view of the degeneracy of the genetic code, considerablesequence variation is possible among these polynucleotide molecules.Thus, the present invention contemplates zB7H6 polypeptide-encodingnucleic acid molecules comprising degenerate nucleotides of SEQ ID NO:1,and their RNA equivalents. The degenerate codons, encompassing allpossible codons for a given amino acid, are set forth in Table 3.

TABLE 3 Amino Acids and Corresponding Degenerate Codons One    AminoLetter Degenerate Acid Code Codons Codon Cys C TGC TGT TGY Ser SAGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT CAN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

With regard to expression in particular host cells, different speciescan exhibit “preferential codon usage.” See generally Grantham et al.,Nucl. Acids Res. 8:1893, 1980; Haas et al. Curr. Biol. 6:315, 1996;Wain-Hobson et al., Gene 13:355, 1981; Grosjean and Fiers, Gene 18:199,1982; Holm, Nuc. Acids Res. 14:3075, 1986; Ikemura, J. Mol. Biol.158:573, 1982; Sharp and Matassi, Curr. Opin. Genet. Dev. 4:851, 1994;Kane, Curr. Opin. Biotechnol. 6:494, 1995; and Makrides, Microbiol. Rev.60:512, 1996. As used herein, the term “preferential codon usage” or“preferential codons” is a term of art referring to protein translationcodons that are most frequently used in cells of a certain species, thusfavoring one or a few representatives of the possible codons encodingeach amino acid (See Table 2). For example, the amino acid threonine(Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cellsACC is the most commonly used codon; in other species, for example,insect cells, yeast, viruses or bacteria, different Thr codons may bepreferential. Preferential codons for a particular species can beintroduced into the polynucleotides of the present invention by avariety of methods known in the art. Introduction of preferential codonsequences into recombinant DNA can, for example, enhance production ofthe protein by making protein translation more efficient within aparticular cell type or species. Therefore, the degenerate codonsequences disclosed herein serve as a template for optimizing expressionof polynucleotides in various cell types and species commonly used inthe art and disclosed herein. Sequences containing preferential codonscan be tested and optimized for expression in various species, andtested for functionality as disclosed herein.

Those skilled in the art will recognize that the sequence disclosed inSEQ ID NO:1 represents a single allele of human zB7H6, and that allelicvariation and alternative splicing are expected to occur. Allelicvariants of this sequence can be cloned by probing cDNA or genomiclibraries from different individuals according to standard procedures.Allelic variants of the nucleotide sequences disclosed herein, includingthose containing silent mutations and those in which mutations result inamino acid sequence changes, are within the scope of the presentinvention, as are proteins which are allelic variants of the amino acidsequences disclosed herein. cDNA molecules generated from alternativelyspliced mRNAs, encoding zB7H6 polypeptides that retain the properties ofthe zB7H6 polypeptide of SEQ ID NO:2 (e.g., variants of theextracellular domain of SEQ ID NO:2 residues 25-266 that retainNKp30-binding capability), are included within the scope of the presentinvention, as are polypeptides encoded by such cDNAs and mRNAs. Allelicvariants and splice variants of these sequences can be cloned by probingcDNA or genomic libraries from different individuals or tissuesaccording to standard procedures known in the art.

Variant zB7H6 nucleic acid molecules can be identified using techniquesgenerally known in the art. Suitable criteria for identification of suchvariants include (a) a determination of the sequence identity orsimilarity between the encoded polypeptide with the amino acid sequenceof SEQ ID NO:2, or a region thereof corresponding to the B7H6extracellular domain of SEQ ID NO:2 residues 25-266; and (b) ahybridization assay. Such zB7H6 nucleic acid variants include nucleicacid molecules that (1) remain hybridized with a nucleic acid moleculehaving the nucleotide sequence of SEQ ID NO:1 (or its complement, or afragment comprising SEQ ID NO:1 residues 73-798) under stringent washingconditions, in which the wash stringency is equivalent to 0.5×-2×SSCwith 0.1% SDS at 55-65° C.; and (2) encode a polypeptide having at least80%, at least 85%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% sequence identity to the amino acidsequence of SEQ ID NO:2, or to residues 25-266 of SEQ ID NO:2.Alternatively, zB7H6 variants can be characterized as nucleic acidmolecules that (1) remain hybridized with a nucleic acid molecule havingthe nucleotide sequence of SEQ ID NO: 1 (or its complement, or afragment comprising SEQ ID NO:1 residues 73-798) under highly stringentwashing conditions, in which the wash stringency is equivalent to0.1×-0.2×SSC with 0.1% SDS at 50-65° C., and (2) encode a polypeptidehaving at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% sequence identity tothe amino acid sequence of SEQ ID NO:2, or to residues 25-266 of SEQ IDNO:2.

In general, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of the target sequencehybridizes to a perfectly matched probe. Following hybridization, thenucleic acid molecules can be washed to remove non-hybridized nucleicacid molecules under stringent conditions, or under highly stringentconditions. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition (Cold Spring Harbor Press 1989); Ausubel et al.,(eds.), Current Protocols in Molecular Biology (John Wiley and Sons,Inc. 1987); Berger and Kimmel (eds.), Guide to Molecular CloningTechniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev. Biochem.Mol. Biol. 26:227, 1990). Sequence analysis software such as OLIGO 6.0(LSR; Long Lake, Minn.) and Primer Premier 4.0 (Premier BiosoftInternational; Palo Alto, Calif.), as well as sites on the Internet, areavailable tools for analyzing a given sequence and calculating T_(m)based on user-defined criteria. It is well within the abilities of oneskilled in the art to adapt hybridization and wash conditions for usewith a particular polynucleotide hybrid.

Percent sequence identity can be readily determined by conventionalmethods such as described supra.

In some embodiments, variants of zB7H6 are characterized by having atleast 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the corresponding aminoacid sequence (e.g., residues 25-266 of SEQ ID NO:2), wherein thevariation in amino acid sequence is due to one or more conservativeamino acid substitutions. Conservative amino acid changes in azB7H6-encoding polynucleotide can be introduced, for example, bysubstituting nucleotides for the nucleotides recited in SEQ ID NO:1.Such “conservative amino acid” variants can be obtained byoligonucleotide-directed mutagenesis, linker-scanning mutagenesis,mutagenesis using the polymerase chain reaction, and the like (seeAusubel (1995); and McPherson (ed.), Directed Mutagenesis: A PracticalApproach (IRL Press 1991)). As noted supra, a functional zB7H6 variantpolypeptide can be identified by the ability to specifically bind toNKp30 (e.g., human NKp30), and/or assays to assess the ability of thevariant or fragment to trigger NKp30-mediated NK cell activation.

The zB7H6 polypeptides of the present invention can also comprisenon-naturally occurring amino acid residues. Non-naturally occurringamino acids include, without limitation, trans-3-methylproline,2,4-methanoproline, cis-4-hydroxyproline, trans-4-hydroxyproline,N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine,hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid,thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline,3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.Several methods are known in the art for incorporating non-naturallyoccurring amino acid residues into proteins. For example, an in vitrosystem can be employed wherein nonsense mutations are suppressed usingchemically aminoacylated suppressor tRNAs. Methods for synthesizingamino acids and aminoacylating tRNA are known in the art. Transcriptionand translation of plasmids containing nonsense mutations is typicallycarried out in a cell-free system comprising an E. coli S30 extract andcommercially available enzymes and other reagents. Proteins are purifiedby chromatography. (See, e.g., Robertson et al., J. Am. Chem. Soc.113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung etal., Science 259:806, 1993; and Chung et al., Proc. Nat'l Acad. Sci. USA90:10145, 1993.) In a second method, translation is carried out inXenopus oocytes by microinjection of mutated mRNA and chemicallyaminoacylated suppressor tRNAs. (See Turcatti et al., J. Biol. Chem.271:19991, 1996.) Within a third method, E. coli cells are cultured inthe absence of a natural amino acid that is to be replaced (e.g.,phenylalanine) and in the presence of the desired non-naturallyoccurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine,4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturallyoccurring amino acid is incorporated into the protein in place of itsnatural counterpart. (See Koide et al., Biochem. 33:7470, 1994.) Also,naturally occurring amino acid residues can be converted tonon-naturally occurring species by in vitro chemical modification.Chemical modification can be combined with site-directed mutagenesis tofurther expand the range of substitutions. (See Wynn and Richards,Protein Sci. 2:395, 1993.)

A limited number of non-conservative amino acids, amino acids that arenot encoded by the genetic code, non-naturally occurring amino acids,and unnatural amino acids may be substituted for zB7H6 amino acidresidues.

Essential amino acids in the polypeptides of the present invention canbe identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis. (See, e.g.,Cunningham and Wells, Science 244:1081, 1989; Bass et al., Proc. Nat'lAcad. Sci. USA 88:4498, 1991; Coombs and Corey, “Site-DirectedMutagenesis and Protein Engineering,” in Proteins: Analysis and Design259-311 (Angeletti, ed., Academic Press, Inc. 1998.) In the lattertechnique, single alanine mutations are introduced at every residue inthe molecule, and the resultant mutant molecules are tested forbiological activity (e.g., NKp30-binding and/or the ability of thevariant or fragment to trigger NKp30-mediated NK cell activation) toidentify amino acid residues that are critical to the activity of themolecule. (See, e.g., Hilton et al., J. Biol. Chem. 271:4699, 1996.)

Although sequence analysis can be used to further define the zB7H6NKp30-binding region, amino acids that play a role in zB7H6 binding toNKp30 can also be determined by physical analysis of structure, asdetermined by such techniques as nuclear magnetic resonance,crystallography, electron diffraction or photoaffinity labeling, inconjunction with mutation of putative contact site amino acids. (See,e.g., de Vos et al., Science 255:306, 1992; Smith et al., J. Mol. Biol.224:899, 1992; and Wlodaver et al., FEBS Lett. 309:59, 1992.)

Multiple amino acid substitutions can be made and tested using knownmethods of mutagenesis and screening, such as those disclosed byReidhaar-Olson and Sauer (Science 241:53, 1988) or Bowie and Sauer(Proc. Nat'l Acad. Sci. USA 86:2152, 1989). Briefly, these authorsdisclose methods for simultaneously randomizing two or more positions ina polypeptide, selecting for functional polypeptide, and then sequencingthe mutagenized polypeptides to determine the spectrum of allowablesubstitutions at each position. Other methods that can be used includephage display (see, e.g., Lowman et al., Biochem. 30:10832, 1991; U.S.Pat. No. 5,223,409 to Ladner et al.; International Publication No. WO92/06204 (Huse)) and region-directed mutagenesis (see, e.g., Derbyshireet al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

Variants zB7H6 nucleotide and polypeptide sequences can also begenerated through DNA shuffling. (See, e.g., Stemmer, Nature 370:389,1994; Stemmer, Proc. Nat'l Acad. Sci. USA 91:10747, 1994; InternationalPublication No. WO 97/20078.) Briefly, variant DNA molecules aregenerated by in vitro homologous recombination by random fragmentationof a parent DNA followed by reassembly using PCR, resulting in randomlyintroduced point mutations. This technique can be modified by using afamily of parent DNA molecules, such as allelic variants or DNAmolecules from different species, to introduce additional variabilityinto the process. Selection or screening for the desired activity,followed by additional iterations of mutagenesis and assay provides forrapid “evolution” of sequences by selecting for desirable mutationswhile simultaneously selecting against detrimental changes.

Mutagenesis methods as disclosed herein can be combined withhigh-throughput, automated screening methods to detect activity ofcloned, mutagenized polypeptides in host cells. Mutagenized DNAmolecules that encode biologically active polypeptides (e.g.,polypeptides that specifically bind NKp30) can be recovered from thehost cells and rapidly sequenced using modern equipment. These methodsallow the rapid determination of the importance of individual amino acidresidues in a polypeptide of interest, and can be applied topolypeptides of unknown structure.

As previously discussed, the present invention also includes “functionalfragments” of the zB7H6 extracellular domain and nucleic acid moleculesencoding such functional fragments. Routine deletion analyses of nucleicacid molecules can be performed to obtain functional fragments of anucleic acid molecule encoding a zB7H6 extracellular domain. As anillustration, DNA molecules having the nucleotide sequence of residues73-798 of SEQ ID NO:1 can be digested with Bal31 nuclease to obtain aseries of nested deletions. The fragments are then inserted intoexpression vectors in proper reading frame, and the expressedpolypeptides are isolated and tested for the ability to bind NKp30. Onealternative to exonuclease digestion is to use oligonucleotide-directedmutagenesis to introduce deletions or stop codons to specify productionof a desired fragment. Alternatively, particular fragments of a zB7H6gene can be synthesized using the polymerase chain reaction.

This general approach is exemplified by studies on the truncation ateither or both termini of interferons. (See Horisberger and Di Marco,Pharmac. Ther. 66:507, 1995.) Moreover, standard techniques forfunctional analysis of proteins are described by, for example, Treuteret al., Molec. Gen. Genet. 240:113, 1993; Content et al., “Expressionand preliminary deletion analysis of the 42 kDa 2-5A synthetase inducedby human interferon,” in Biological Interferon Systems, Proceedings ofISIR-TNO Meeting on Interferon Systems 65-72 (Cantell, ed., Nijhoff1987); Herschman, “The EGF Receptor,” in Control of Animal CellProliferation, Vol. 1 169-199 (Boynton et al., eds., Academic Press1985); Coumailleau et al., J. Biol. Chem. 270:29270, 1995; Fukunaga etal., J. Biol. Chem. 270:25291, 1995; Yamaguchi et al., Biochem.Pharmacol. 50:1295, 1995; and Meisel et al., Plant Molec. Biol. 30:1,1996.

The present invention also includes functional fragments of a zB7H6polynucleotide encoding a polypeptide that has amino acid changesrelative to the amino acid sequence of SEQ ID NO:2 (e.g., changesrelative to residues 25-266 of SEQ ID NO:2). A variant zB7H6 gene can beidentified on the basis of structure by determining the level ofidentity with disclosed nucleotide and amino acid sequences, asdiscussed above. An alternative approach to identifying a variant geneon the basis of structure is to determine whether a nucleic acidmolecule encoding a potential variant zB7H6 gene can hybridize to anucleic acid molecule comprising a nucleotide sequence, such as SEQ IDNO:1.

Polynucleotide molecules comprising a polynucleotide sequence providedherein are propagated by placing the molecule in a vector. Viral andnon-viral vectors are used, including plasmids. The choice of plasmidwill depend on the type of cell in which propagation is desired and thepurpose of propagation. Certain vectors are useful for amplifying andmaking large amounts of the desired DNA sequence. Other vectors aresuitable for expression in cells in culture. Still other vectors aresuitable for transfer and expression in cells in a whole animal orperson. The choice of appropriate vector is well within the skill of theart. Many such vectors are available commercially. The partial orfull-length polynucleotide is inserted into a vector typically by meansof DNA ligase attachment to a cleaved restriction enzyme site in thevector. Alternatively, the desired nucleotide sequence can be insertedby homologous recombination in vivo. Typically this is accomplished byattaching regions of homology to the vector on the flanks of the desirednucleotide sequence. Regions of homology are added by ligation ofoligonucleotides, or by polymerase chain reaction using primerscomprising both the region of homology and a portion of the desirednucleotide sequence, for example.

For expression, an expression cassette or system may be employed. Toexpress a zB7H6 gene, a nucleic acid molecule encoding the polypeptide,operably linked to regulatory sequences that control transcriptionalexpression in an expression vector, is introduced into a host cell. Inaddition to transcriptional regulatory sequences, such as promoters andenhancers, expression vectors can include translational regulatorysequences and a marker gene which is suitable for selection of cellsthat carry the expression vector. The gene product encoded by apolynucleotide of the invention is expressed in any convenientexpression system, including, for example, bacterial, yeast, insect,amphibian and mammalian systems. Suitable vectors and host cells aredescribed, e.g., in U.S. Pat. No. 5,654,173. In the expression vector,the zB7H6 polypeptide-encoding polynucleotide is linked to a regulatorysequence as appropriate to obtain the desired expression properties.These can include promoters (attached either at the 5′ end of the sensestrand or at the 3′ end of the antisense strand), enhancers,terminators, operators, repressors, and inducers. The promoters can beregulated or constitutive. In some situations it may be desirable to useconditionally active promoters, such as tissue-specific or developmentalstage-specific promoters. These are linked to the desired nucleotidesequence using the techniques described above for linkage to vectors.Any techniques known in the art can be used. Accordingly, the expressionvector will generally provide a transcriptional and translationalinitiation region, which may be inducible or constitutive, where thecoding region is operably linked under the transcriptional control ofthe transcriptional initiation region, and a transcriptional andtranslational termination region. These control regions may be native tothe DNA encoding the zB7H6 polypeptide or may be derived from exogenoussources.

The expression cassettes may be introduced into a variety of vectors,e.g., plasmid, BAC, YAC, bacteriophage such as lambda, P1, M13, etc.,plant or animal viral vectors (e.g., retroviral-based vectors,adenovirus vectors), and the like, where the vectors are normallycharacterized by the ability to provide selection of cells comprisingthe expression vectors. The vectors may provide for extrachromosomalmaintenance, particularly as plasmids or viruses, or for integrationinto the host chromosome. Where extrachromosomal maintenance is desired,an origin sequence is provided for the replication of the plasmid, whichmay be low- or high copy-number. A wide variety of markers are availablefor selection, particularly those which protect against toxins, moreparticularly against antibiotics. The particular marker that is chosenis selected in accordance with the nature of the host, where in somecases, complementation may be employed with auxotrophic hosts.Introduction of the DNA construct may use any convenient method,including, e.g., conjugation, bacterial transformation,calcium-precipitated DNA, electroporation, fusion, transfection,infection with viral vectors, biolistics, and the like.

zB7H6 polypeptides may be expressed in prokaryotes or eukaryotes inaccordance with conventional ways, depending upon the purpose forexpression. For large scale production of the protein, a unicellularorganism, such as E. coli, B. subtilis, S. cerevisiae, insect cells incombination with baculovirus vectors, or cells of a higher organism suchas vertebrates, particularly mammals (e.g., COS 7 cells, HEK 293, CHO,Xenopus Oocytes), may be used as the expression host cells. Accordingly,specific expression systems of interest include bacterial, yeast, insectcell and mammalian cell derived expression systems. Representativeexpression systems in bacteria include, e.g., those described in Changet al., Nature 275:615, 1978; Goeddel et al., Nature (1979) 281:544,1979; Goeddel et al., Nucleic Acids Res. 8:4057, 1980; EP 0 036,776;U.S. Pat. No. 4,551,433; DeBoer et al., Proc. Natl. Acad. Sci. USA80:21-25, 1983; and Siebenlist et al., Cell 20:269, 1980. Representativeexpression systems in yeast include, e.g., those described in Hinnen etal., Proc. Natl. Acad. Sci. USA 75:1929, 1978; Ito et al., J. Bacteriol.153:163, 1983; Kurtz et al., Mol. Cell. Biol. 6:142, 1986; Kunze et al.,J. Basic Microbiol. 25:141, 1985; Gleeson et al., J. Gen. Microbiol.132:3459, 1986; Roggenkamp et al., Mol. Gen. Genet. 202:302, 1986; Daset al., J. Bacteriol. 158:1165, 1984; De Louvencourt et al., J.Bacteriol. 154:737, 1983; Van den Berg et al., Bio/Technology 8:135,1990; Kunze et al., J. Basic Microbiol. 25:141, 1985; Cregg et al., Mol.Cell. Biol. 5:3376, 1985; U.S. Pat. Nos. 4,837,148 and 4,929,555; Beachand Nurse, Nature 300:706, 1981; Davidow et al., Curr. Genet. 10:380,1985; Gaillardin et al., Curr. Genet. 10:49, 1985; Ballance et al.,Biochem. Biophys. Res. Commun. 112:284-289, 1983; Tilburn et al., Gene26:205-221, 1983; Yelton et al., Proc. Natl. Acad. Sci. USA81:1470-1474, 1984; Kelly and Hynes, EMBO J. 4:475479, 1985; EP 0244,234; and WO 91/00357. Representative expression systems in insectcells include, e.g., those described in U.S. Pat. No. 4,745,051; Friesenet al., “The Regulation of Baculovirus Gene Expression”, in: TheMolecular Biology Of Baculoviruses (W. Doerfler, ed., 1986); EP 0127,839; EP 0 155,476; and Vlak et al., J. Gen. Virol. 69:765-776, 1988;Miller et al., Ann. Rev. Microbiol. 42:177, 1988; Carbonell et al., Gene73:409, 1988; Maeda et al., Nature 315:592-594, 1985; Lebacq-Verheydenet al., Mol. Cell. Biol. 8:3129, 1988; Smith et al., Proc. Natl. Acad.Sci. USA 82:8844, 1985; Miyajima et al., Gene 58:273, 1987; and Martinet al., DNA 7:99, 1988. Numerous baculoviral strains and variants andcorresponding permissive insect host cells from hosts are described inLuckow et al., Bio/Technology 6:47-55, 1988; Miller et al., GenericEngineering 8:277-279, 1986; and Maeda et al., Nature 15:592-594, 1985.Representative expression systems in mammalian cells include, e.g.,those described in Dijkema et al., EMBO J. 4:761, 1985, Gorman et al.,Proc. Natl. Acad. Sci. USA 79:6777, 1982; Boshart et al., Cell 41:521,1985; and U.S. Pat. No. 4,399,216. Other features of mammalianexpression are facilitated, for example, as described in Ham andWallace, Meth. Enz. 58:44, 1979; Barnes and Sato, Anal. Biochem.102:255, 1980; U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762,4,560,655, WO 90/103430, WO 87/00195, and U.S. Pat. No. RE 30,985.

The subject nucleic acids can be used to generate genetically modifiednon-human animals or site specific gene modifications in cell lines. Theterm “transgenic” is intended to encompass genetically modified animalshaving the addition of DNA encoding the zB7H6 polypeptide or having anexogenous DNA encoding the zB7H6 polypeptide that is stably transmittedin the host cells. Transgenic animals may be made through homologousrecombination. Alternatively, a nucleic acid construct is randomlyintegrated into the genome. Vectors for stable integration includeplasmids, retroviruses and other animal viruses, YACs, and the like. Ofinterest are transgenic mammals, particularly rodents (e.g., rats,mice).

DNA constructs for homologous recombination will comprise at least aportion of the DNA encoding the soluble zB7H6 polypeptide and willinclude regions of homology to the target locus. Conveniently, markersfor positive and negative selection are included. Methods for generatingcells having targeted gene modifications through homologousrecombination are known in the-art. For various techniques fortransfecting mammalian cells, see, for example, Known et al. Methods inEnzymology 185:527-537, 1990.

For embryonic stem (ES) cells, an ES cell line may be employed, or EScells may be obtained freshly from a host (e.g., mouse, rat, guineapig). Such cells are grown on an appropriate fibroblast-feeder layer orgrown in the presence of leukemia inhibiting factor (LIF). When ES cellshave been transformed, they may be used to produce transgenic animals.After transformation, the cells are plated onto a feeder layer in anappropriate medium. Cells containing the construct may be detected byemploying a selective medium. After sufficient time for colonies togrow, they are picked and analyzed for the occurrence of homologousrecombination. Those colonies that show homologous recombination maythen be used for embryo manipulation and blastocyst injection.Blastocysts are obtained from 4 to 6 week old superovulated females. TheES cells are trypsinized, and the modified cells are injected into theblastocoel of the blastocyst. After injection, the blastocysts arereturned to each uterine horn of pseudopregnant females. Females arethen allowed to go to term and the resulting litters screened for mutantcells having the construct. By providing for a different phenotype ofthe blastocyst and the ES cells, chimeric progeny can be readilydetected. The chimeric animals are screened for the presence of the DNAencoding the zB7H6 polypeptide and males and females having themodification are mated to produce homozygous progeny. The transgenicanimals may be any non-human mammal, such as, e.g., laboratory animalsor domestic animals. The transgenic animals may be used to determine theeffect of a candidate drug in an in vivo environment.

The present invention further includes the recombinant vectors and hostcells comprising the vectors as described herein. In general,recombinant vectors and host cells of the invention are isolated;however, a host cell comprising a polynucleotide of the invention may bepart of a genetically modified animal.

When any of the above host cells, or other appropriate host cells ororganisms, are used to replicate and/or express the polynucleotides ornucleic acids of the invention, the resulting replicated nucleic acid,RNA, expressed protein or polypeptide, is within the scope of theinvention as a product of the host cell or organism. The product isrecovered by any appropriate means known in the art. zB7H6 polypeptidescan be produced as monomers or multimer (e.g., homodimers, heterodimers,tetramers)

Accordingly, in yet another aspect, the present invention provides amethod of preparing a soluble zB7H6 polypeptide, including monomeric andmultimeric (e.g., homodimeric, heterodimeric, tetrameric) forms thereof,using recombinant host cells as described herein. Such methods generallyinclude culturing a host cell transformed or transfected with anexpression vectors encoding the soluble zB7H6 protein under conditionsin which the protein in expressed, and recovering the soluble zB7H6protein from the host cell. Techniques for recovering recombinantproteins for prokaryotic and eukaryotic host cells are generallywell-known in the art.

For example, general methods for expressing and recovering foreignprotein produced by a mammalian cell system are provided by, forexample, Etcheverry, “Expression of Engineered Proteins in MammalianCell Culture,” in Protein Engineering: Principles and Practice 163(Cleland et al., eds., Wiley-Liss, Inc. 1996). Standard techniques forrecovering protein produced by a bacterial system are provided by, forexample, Grisshammer et al., “Purification of over-produced proteinsfrom E. coli cells,” in DNA Cloning 2: Expression Systems, 2nd Edition59-92 (Glover et al., eds., Oxford University Press 1995). Establishedmethods for isolating recombinant proteins from a baculovirus system aredescribed by, e.g., Richardson (ed.), Baculovirus Expression Protocols(The Humana Press, Inc. 1995).

When expressing a soluble zB7H6 polypeptide in bacteria such as E. coli,the polypeptide may be retained in the cytoplasm, typically as insolublegranules, or may be directed to the periplasmic space by a bacterialsecretion sequence. In the former case, the cells are lysed, and thegranules are recovered and denatured using, for example, guanidineisothiocyanate or urea. The denatured polypeptide can then be refoldedand dimerized by diluting the denaturant, such as by dialysis against asolution of urea and a combination of reduced and oxidized glutathione,followed by dialysis against a buffered saline solution. In the lattercase, the polypeptide can be recovered from the periplasmic space in asoluble and functional form by disrupting the cells (by, for example,sonication or osmotic shock) to release the contents of the periplasmicspace and recovering the protein, thereby obviating the need fordenaturation and refolding.

Alternatively, zB7H6 polypeptides of the present invention can besynthesized by exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis. Thesesynthesis methods are well-known to those of skill in the art. (See,e.g., Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al.,“Solid Phase Peptide Synthesis” (2nd ed., Pierce Chemical Co. 1984);Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; Atherton et al., SolidPhase Peptide Synthesis: A Practical Approach (IRL Press 1989); Fieldsand Colowick, “Solid-Phase Peptide Synthesis,” Methods in EnzymologyVolume 289 (Academic Press 1997); and Lloyd-Williams et al., ChemicalApproaches to the Synthesis of Peptides and Proteins (CRC Press, Inc.1997).) Variations in total chemical synthesis strategies, such as“native chemical ligation” and “expressed protein ligation” are alsostandard. (See, e.g., Dawson et al., Science 266:776, 1994; Hackeng etal., Proc. Nat'l Acad. Sci. USA 94:7845, 1997; Dawson, Methods Enzymol.287:34, 1997; Muir et al, Proc. Nat'l Acad. Sci. USA 95:6705, 1998; andSeverinov and Muir, J. Biol. Chem. 273:16205, 1998.)

As previously discussed, soluble zB7H6 polypeptides can be producedeither as monomers or in any of various multimeric forms (e.g.,homodimers, heterodimers, or tetramers). In the case of recombinantlyproduced heteromultimers, comprising at least one polypeptide chain thatis a soluble zB7H6 polypeptide as described herein and at least oneother polypeptide chain that is a soluble non-zB7H6 polypeptide, hostcells are transformed or transfected with different expression vectorsencoding the different polypeptide chains. In some embodiments, the samehost cell is transfected or transformed with different expressionvectors encoding the different chains of a heteromultimer andheteromultimeric protein is then isolated from the medium;alternatively, each vector encoding a different polypeptide chain can beseparately produced in different host cell populations and subsequentlyused to form multimeric complexes following isolation of recombinantprotein. For example, different polypeptide chain components can becombined in deliberate ratios to result in the heteromultimericmolecules desired. Different polypeptide chains of a heteromultimer canbe differentially labeled with various tag sequences (e.g., His tag,FLAG tag, and Glu-Glu tag) to allow analysis of the composition orpurification of the resulting molecules. In particular embodiments, theheteromultimer is a heterodimer (such as, e.g., a dimer in which onepolypeptide chain is a soluble zB7H6 fusion protein comprising, forexample, an immunoglobulin heavy chain region) or a heterotetramer (suchas, e.g., a tetramer in which at least one polypeptide chain is asoluble zB7H6 fusion protein comprising, e.g., a VASP domain).

The polypeptides of the present invention are typically purified to atleast about 80% purity, more typically to at least about 90% purity andpreferably to at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% purity with respect tocontaminating macromolecules, particularly other proteins and nucleicacids, and free of infectious and pyrogenic agents. The polypeptides ofthe present invention may also be purified to a pharmaceutically purestate, which is greater than 99.9% pure. In certain preparations,purified polypeptide is substantially free of other polypeptides,particularly other polypeptides of animal origin.

Fractionation and/or conventional purification methods can be used toobtain zB7H6 polypeptide preparations purified from natural sources(e.g., human tissue sources), synthetic zB7H6 polypeptides, andrecombinant zB7H6 polypeptides purified from recombinant host cells. Ingeneral, ammonium sulfate precipitation and acid or chaotrope extractionmay be used for fractionation of samples. Exemplary purification stepsmay include hydroxyapatite, size exclusion, FPLC and reverse-phase highperformance liquid chromatography. Suitable chromatographic mediainclude derivatized dextrans, agarose, cellulose, polyacrylamide,specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives aresuitable. Exemplary chromatographic media include those mediaderivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties.

Examples of coupling chemistries include cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, hydrazide activation, and carboxyl and amino derivatives forcarbodiimide coupling chemistries. These and other solid media are wellknown and widely used in the art, and are available from commercialsuppliers. Selection of a particular method for polypeptide isolationand purification is a matter of routine design and is determined in partby the properties of the chosen support. See, e.g., AffinityChromatography: Principles &Methods (Pharmacia LKB Biotechnology 1988);and Doonan, Protein Purification Protocols (The Humana Press 1996).

Additional variations in zB7H6 polypeptide isolation and purificationcan be devised by those of skill in the art. For example, anti-zB7H6antibodies, obtained as described below, can be used to isolate largequantities of protein by immunoaffinity purification.

The polypeptides of the present invention can also be isolated byexploitation of particular properties. For example, immobilized metalion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1, 1985). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography and ion exchange chromatography (see,e.g., M. Deutscher, (ed.), Meth. Enzymol. 182:529, 1990). Withinadditional embodiments of the invention, a fusion of the polypeptide ofinterest and an affinity tag (e.g., maltose-binding protein, animmunoglobulin domain) may be constructed to facilitate purification.Moreover, the counter-receptor-binding properties of zB7H6 extracellulardomain can be exploited for purification of zB7H6 polypeptides; forexample, by using affinity chromatography wherein NKp30 is bound to acolumn and the zB7H6 polypeptide is bound and subsequently eluted usingstandard chromatography methods.

zB7H6 polypeptides or fragments thereof may also be prepared throughchemical synthesis, as described above. zB7H6 polypeptides may bemonomers or multimers; glycosylated or non-glycosylated; PEGylated ornon-PEGylated; and may or may not include an initial methionine aminoacid residue.

Once produced, function of a zB7H6 polypeptide can be readily assessedusing routine assays. Binding of a zB7H6 polypeptide to NKp30 is onemeasure of functional activity. Such binding activity may be determined,for example, by competition for binding to the binding domain of NKp30(i.e., competitive binding assays). For example, one configuration of acompetitive binding assay uses a labeled, soluble NKp30 receptor (e.g.,a fusion protein comprising the extracellular domain of NKp30 and an Fcfragment conjugated to biotin) and intact cells expressing a native formof zB7H6 (e.g., a polypeptide having the amino acid sequence of SEQ IDNO:2). Such an assay is described in Example 7. Also, binding of solublezB7H6 polypeptides to NKp30-expressing cells may be measured.Alternatively, instead of using soluble zB7H6 or intact cells expressinga native form of zB7H6, one could substitute purified zB7H6 bound to asolid phase. Competitive binding assays can be performed using standardmethodology. Qualitative or semi-quantitative results can be obtained bycompetitive autoradiographic plate binding assays, or fluorescenceactivated cell sorting, or Scatchard plots may be utilized to generatequantitative results.

Function of a zB7H6 polypeptide may also be measured using bioassaysthat measure, e.g., biological activity associated with NKp30 function,including, for example, NK cell cytolytic assays. For example, as shownherein, certain cell lines, such as P815, do not serve as good cytolytictargets for NK-92 cells, which express NKp30. (See, e.g., Example 7.)Expression of hzB7H6 (SEQ ID NO:2) in these cells, however, such as bytransfection with a zB7H6 expression vector, renders the cellsvulnerable to attack by NK-92 cells. (See id.) Accordingly, zB7H6polypeptides, zB7H6 polypeptides having one or more amino acidsubstitutions, addition, or deletions in the extracellular domain, canbe readily screened for functional activity by expressing suchpolypeptides in P815 cells and determining, using NK-92 cells inwell-known cytolytic assays, whether such cells are vulnerable to NKcell attack. An exemplary NK-92 cell assay that can be used to evaluatezB7H6 polypeptide function is described in Example 7, infra.

Other assays for evaluating function of zB7H6 polypeptides include, forexample, addition of a soluble zB7H6 polypeptide to NKp30-expressing NKcells to test for activation of NK cell function against target cells(e.g., P815). NK cell assays for evaluation of antibodies against NKcell-surface receptors have been described, e.g., by Pende et. al. (J.Exp. Med. 190:1505-1516, 1999), and such assays are readily amenable toadaptation for evaluating activity of soluble zB7H6 polypeptides asdescribed herein.

IV. Antibodies to zB7H6 Proteins

In another aspect, the present invention provides antibodies thatspecifically bind to zB7H6. In preferred embodiments, an anti-zB7H6antibody of the invention is an isolated antibody that specificallybinds to an extracellular domain of zB7H6 (e.g., to a polypeptidesegment having the amino acid sequence set forth in residues 25-266 ofSEQ ID NO:2). In some embodiments, an anti-zB7H6 antibody of theinvention is capable of inhibiting the interaction of zB7H6 with humanNKp30; such antibodies are useful, for example, for inhibiting cellularor other physiological events associated with the interaction of zB7H6with NKp30, including, for example, zB7H6- and/or NKp30-mediatedintracellular signaling and associated effector function (e.g.,NKp30-mediated cytolytic activity).

Antibodies to zB7H6 can be obtained, for example, using the product of azB7H6 expression vector or zB7H6 isolated from a natural source as anantigen. Particularly useful anti-zB7H6 antibodies “bind specifically”to zB7H6. Antibodies are considered to be specifically binding if theantibodies exhibit at least one of the following two properties: (1)antibodies bind to zB7H6 with a threshold level of binding activity, and(2) antibodies do not significantly cross-react with polypeptidesrelated to zB7H6.

With regard to the first characteristic, antibodies specifically bind ifthey bind to a zB7H6 polypeptide, peptide, or epitope with a bindingaffinity (K_(a)) of 10⁶ M⁻¹ or greater, preferably 10⁷ M⁻¹ or greater,more preferably 10⁸ M⁻¹ or greater, and most preferably 10⁹ M⁻¹ orgreater. The binding affinity of an antibody can be readily determinedby one of ordinary skill in the art, for example, by Scatchard analysis(Scatchard, Ann. NY Acad. Sci. 51:660, 1949). With regard to the secondcharacteristic, antibodies do not significantly cross-react with relatedpolypeptide molecules, for example, if they detect zB7H6, but notpresently known polypeptides using a standard Western blot analysis.Examples of known related polypeptides include known B7 family members.

Anti-zB7H6 antibodies can be produced using antigenic zB7H6epitope-bearing peptides and polypeptides. Antigenic epitope-bearingpeptides and polypeptides typically contain a sequence of at least nine,or between 15 to about 30 amino acids contained within the amino acidsequence of SEQ ID NO:2. However, peptides or polypeptides comprising alarger portion of an amino acid sequence of the invention, containingfrom 30 to 50 amino acids, or any length up to and including the entireamino acid sequence of a zB7H6 polypeptide, also are useful for inducingantibodies that bind with zB7H6. It is desirable that the amino acidsequence of the epitope-bearing peptide is selected to providesubstantial solubility in aqueous solvents (i.e., the sequence includesrelatively hydrophilic residues, while hydrophobic residues aretypically avoided). In addition, amino acid sequences containing prolineresidues may be also be desirable for antibody production.

Potential antigenic sites in zB7H6 can be identified using theJameson-Wolf method, Jameson and Wolf (CABIOS 4:181, 1988), asimplemented by the PROTEAN program (version 3.14) of LASERGENE (DNASTAR;Madison, Wis.). Default parameters may be used in this analysis.

The Jameson-Wolf method predicts potential antigenic determinants bycombining six major subroutines for protein structural prediction. Forexample, the Hopp-Woods method (see Hopp et al., Proc. Nat'l Acad. Sci.USA 78:3824, 1981) may first be used to identify amino acid sequencesrepresenting areas of greatest local hydrophilicity (parameter: sevenresidues averaged). In the second step, Emini's method (see Emini etal., J. Virology 55:836, 1985) may be used to calculate surfaceprobabilities (parameter: surface decision threshold (0.6)=1). Third,the Karplus-Schultz method, Karplus and Schultz (Naturwissenschaften72:212, 1985) may be used to predict backbone chain flexibility(parameter: flexibility threshold (0.2)=1). In fourth and fifth steps ofanalysis, secondary structure predictions may be applied to the datausing the methods of Chou-Fasman (see Chou, “Prediction of ProteinStructural Classes from Amino Acid Composition,” in Prediction ofProtein Structure and the Principles of Protein Conformation 549-586(Fasman, ed., Plenum Press 1990) and Garnier-Robson (see Garnier et al.,J. Mol. Biol. 120:97, 1978) (Chou-Fasman parameters: conformationtable=64 proteins; α region threshold=103; β region threshold=105;Garnier-Robson parameters: α and β decision constants=0). In a sixthsubroutine, flexibility parameters and hydropathy/solvent accessibilityfactors may be combined to determine a surface contour value, designatedas the “antigenic index.” Finally, a peak broadening function may beapplied to the antigenic index, which broadens major surface peaks byadding, e.g., 20, 40, 60, or 80% of the respective peak value to accountfor additional free energy derived from the mobility of surface regionsrelative to interior regions. This calculation, however, is typicallynot applied to any major peak that resides in a helical region, sincehelical regions tend to be less flexible.

Polyclonal antibodies to recombinant zB7H6 protein or to zB7H6 isolatedfrom natural sources can be prepared using methods well-known to thoseof skill in the art. (See, e.g., Green et al., “Production of PolyclonalAntisera,” in Immunochemical Protocols 1-5 (Manson, ed., Humana Press1992); Williams et al., “Expression of foreign proteins in E. coli usingplasmid vectors and purification of specific polyclonal antibodies,” inDNA Cloning 2: Expression Systems, 2nd Edition 15 (Glover et al., eds.,Oxford University Press 1995). The immunogenicity of a zB7H6 polypeptidecan be increased through the use of an adjuvant, such as alum (aluminumhydroxide) or Freund's complete or incomplete adjuvant. Polypeptidesuseful for immunization also include fusion polypeptides, such asfusions of zB7H6 or a portion thereof with an immunoglobulin polypeptideor with maltose binding protein. The polypeptide immunogen may be afull-length molecule or a portion thereof. If the polypeptide portion is“hapten-like,” for immunization, such portion may be advantageouslyjoined or linked to a macromolecular carrier such as, for example,keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), or tetanustoxoid.

Although polyclonal antibodies are typically raised in animals such ashorses, cows, dogs, chicken, rats, mice, rabbits, guinea pigs, goats, orsheep, an anti-zB7H6 antibody of the present invention may also bederived from a subhuman primate antibody. General techniques for raisingdiagnostically and therapeutically useful antibodies in baboons may befound, for example, in Goldenberg et al., International PatentPublication No. WO 91/11465, and in Losman et al., Int. J. Cancer46:310, 1990.

Alternatively, monoclonal anti-zB7H6 antibodies can be generated. Forexample, rodent monoclonal antibodies to specific antigens may beobtained by methods known to those skilled in the art (see, e.g., Kohleret al., Nature 256:495, 1975; Coligan et al. (eds.), Current Protocolsin Immunology, Vol. 1 2.5.1-2.6.7 (John Wiley & Sons 1991) [“Coligan”];Picksley et al., “Production of monoclonal antibodies against proteinsexpressed in E. coli,” in DNA Cloning 2: Expression Systems, 2nd Edition93 (Glover et al., eds., Oxford University Press 1995). In certainvariations, monoclonal antibodies are obtained by injecting mice with acomposition comprising a zB7H6 gene product (e.g., a polypeptidecomprising or consisting of SEQ ID NO:2 residues 25-266), verifying thepresence of antibody production by removing a serum sample, removing thespleen to obtain B-lymphocytes, fusing the B-lymphocytes with myelomacells to produce hybridomas, cloning the hybridomas, selecting positiveclones which produce antibodies to the antigen, culturing the clonesthat produce antibodies to the antigen, and isolating the antibodiesfrom the hybridoma cultures.

An anti-zB7H6 antibody may also be a human monoclonal antibody, or anantibody derived therefrom. Human monoclonal antibodies are obtainedfrom transgenic mice that have been engineered to produce specific humanantibodies in response to antigenic challenge. In this technique,elements of the human heavy and light chain locus are introduced intostrains of mice derived from embryonic stem cell lines that containtargeted disruptions of the endogenous heavy chain and light chain loci.The transgenic mice can synthesize human antibodies specific for humanantigens, and the mice can be used to produce human antibody-secretinghybridomas. Methods for obtaining human antibodies from transgenic miceare described, for example, by Green et al., Nature Genet. 7:13, 1994;Lonberg et al., Nature 368:856, 1994; and Taylor et al., Int. Immun.6:579, 1994.

Monoclonal antibodies can be isolated and purified from hybridomacultures by a variety of well-established techniques. Such isolationtechniques include affinity chromatography with Protein-A Sepharose,size-exclusion chromatography, and ion-exchange chromatography (see,e.g., Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines etal., “Purification of Immunoglobulin G (IgG),” in Methods in MolecularBiology (Vol. 10) 79-104 (The Humana Press, Inc. 1992)).

In some embodiments, an anti-B7H6 antibody is an antibody fragmentcomprising an antigen-binding domain of an intact (whole) antibody. Suchantibody fragments can be obtained, for example, by proteolytichydrolysis of an antibody. Antibody fragments can be obtained by pepsinor papain digestion of whole antibodies by conventional methods. As anillustration, antibody fragments can be produced by enzymatic cleavageof antibodies with pepsin to provide a 5S fragment denoted F(ab′)₂. Thisfragment can be further cleaved using a thiol reducing agent to produce3.5S Fab′ monovalent fragments. Optionally, the cleavage reaction can beperformed using a blocking group for the sulfhydryl groups that resultfrom cleavage of disulfide linkages. As an alternative, an enzymaticcleavage using pepsin produces two monovalent F_(ab) fragments and anF_(c) fragment directly. These methods are described, for example, inU.S. Pat. No. 4,331,647 to Goldenberg; Nisonoff et al., Arch Biochem.Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al.,in Methods in Enzymology (Vol. 1) 422 (Academic Press 1967); and Coliganat pages 2.8.1-2.8.10 and 2.10.-2.10.4.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association can be noncovalent, as described by Inbar etal., Proc. Nat'l Acad. Sci. USA 69:2659, 1972. Alternatively, thevariable chains can be linked by an intermolecular disulfide bond orcross-linked by chemicals such as glutaraldehyde (see, e.g., Sandhu,Crit. Rev. Biotech. 12:437, 1992).

The Fv fragments may comprise V_(H) and V_(L) chains which are connectedby a peptide linker. These single-chain antigen binding proteins (scFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains which are connected by anoligonucleotide. The structural gene is inserted into an expressionvector which is subsequently introduced into a host cell, such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingscFvs are described, for example, by Whitlow et al., Methods: ACompanion to Methods in Enzymology 2:97, 1991. (See also Bird et al.,Science 242:423, 1988; U.S. Pat. No. 4,946,778 to Ladner et al.; Pack etal., Bio/Technology 11:1271, 1993, and Sandhu, supra.) As anillustration, a scFV can be obtained by exposing lymphocytes to zB7H6polypeptide in vitro, and selecting antibody display libraries in phageor similar vectors (for instance, through use of immobilized or labeledzB7H6 protein or peptide).

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells (see, e.g., Larrick et al.,Methods: A Companion to Methods in Enzymology 2:106, 1991;Courtenay-Luck, “Genetic Manipulation of Monoclonal Antibodies,” inMonoclonal Antibodies: Production, Engineering and Clinical Application166 (Ritter et al., eds., Cambridge University Press 1995); and Ward etal., “Genetic Manipulation and Expression of Antibodies,” in MonoclonalAntibodies: Principles and Applications 137 (Birch et al., eds.,Wiley-Liss, Inc. 1995)).

Alternatively, an anti-zB7H6 antibody may be derived from a “humanized”monoclonal antibody. Humanized monoclonal antibodies are produced bytransferring mouse complementary determining regions from heavy andlight variable chains of the mouse immunoglobulin into a human variabledomain. Typical residues of human antibodies are then substituted in theframework regions of the murine counterparts. The use of antibodycomponents derived from humanized monoclonal antibodies obviatespotential problems associated with the immunogenicity of murine constantregions. General techniques for cloning murine immunoglobulin variabledomains are described, for example, by Orlandi et al., Proc. Nat'l Acad.Sci. USA 86:3833, 1989. Techniques for producing humanized monoclonalantibodies are described, for example, by Jones et al., Nature 321:522,1986; Carter et al., Proc. Nat'l Acad. Sci. USA 89:4285, 1992; Sandhu,Crit. Rev. Biotech. 12:437, 1992; Singer et al., J. Immun. 150:2844,1993; Sudhir (ed.), Antibody Engineering Protocols (Humana Press, Inc.1995); Kelley, “Engineering Therapeutic Antibodies,” in ProteinEngineering Principles and Practice 399-434 (Cleland et al., eds., JohnWiley & Sons, Inc. 1996); and U.S. Pat. No. 5,693,762 to Queen et al.

In certain variations, an anti-zB7H6 antibody includes an Fc region,which comprises the C_(H)2 and C_(H)3 domains of an immunoglobulin (Ig)heavy chain and typically a portion of an Ig hinge region. Fc isresponsible for two of the highly desirable properties of an IgG:recruitment of effector function and a long serum half-life. The abilityto kill target cells to which an antibody is attached stems from theactivation of immune effector pathway (ADCC) and the complement pathway(CDC) through the binding of Fc to Fc receptors and the complementprotein, C1q, respectively. The binding is mediated by residues locatedprimarily in the lower hinge region and upper C_(H)2 domain. (See, e.g.,Wines et al., J. Immunol. 164:5313, 2000; Woof and Burton, NatureReviews 4:1, 2004.) The long half-life in serum demonstrated by IgG ismediated through a pH dependent interaction between amino acids in theC_(H)2 and C_(H)3 domain and the neonatal Fc receptor, FcRn. (See, e.g.,Getie and Ward, Immunology Today 18:592, 1997; Petkova et al., Int.Immunol. 18:1759, 2006.)

Accordingly, in certain embodiments of an anti-zB7H6 antibody comprisingan Fc region, the Fc region has ADCC and/or CDC activity. Suchantibodies are particularly useful for mediating killing of target cellsexpressing zB7H6 such as, for example, cancer cells or virally infectedcells. In other embodiments, an anti-zB7H6 antibody comprises an Fcregion that lacks one or more effector functions (e.g., lacks ADCCand/or CDC activity). Fc regions lacking or having substantially reducedeffector function may be obtained, for example, by introducing one ormore amino acid substitutions into a native Fc region sequence, suchthat the Fc region does not bind, or has substantially reduced binding,to cytolytic Fc receptors and/or the C1q complement protein.Particularly suitable Fc regions lacking or having substantially reducedeffector function include, for example, Fc4 (SEQ ID NO:31), Fc5 (SEQ IDNO:32), and Fc6 (SEQ ID NO:33), and Fc7 (SEQ ID NO:34), which are shownin FIGS. 13A-13C.

In certain embodiments comprising an Fc region, the Fc region is asingle chain Fc (scFc), which comprises two Fc domain monomers joined bya flexible linker, such that the two Fc monomers are capable ofdimerization to form a functional, dimeric Fc domain. For example, insome variations of an anti-zB7H6 antibody comprising a scFc, theantibody comprises a single chain Fv (scFv) fused to the scFc portion,wherein the scFv portion specifically binds to zB7H6. Single chain Fcpolypeptides, including fusion polypeptides comprising scFc and one moreantigen-binding domains (e.g., scFv), are further described inInternational PCT Patent Application No. US08/060,852, entitled “SingleChain Fc, Methods of Making, and Methods of Treatment,” filed Apr. 18,2008, the disclosure of which is incorporated by reference herein in itsentirety.

Moreover, anti-zB7H6 antibodies or antibody fragments of the presentinvention can be PEGylated using methods in the art and describedherein.

Anti-idiotypic antibodies may be raised against an anti-zB7H6 antibodyspecific for the zB7H6 extracellular domain (e.g., against SEQ ID NO:2residues 25-266). In some variations, an anti-idiotype antibody isagainst an anti-zB7H6 antibody that is capable of inhibiting theinteraction of zB7H6 with human NKp30; such anti-idiotype antibodies maymimic the ability of zB7H6 to bind NKp30 and, in preferred embodiments,are capable of triggering or enhancing NKp30-mediated NK cellactivation. Polyclonal anti-idiotype antibodies can be prepared byimmunizing animals with anti-zB7H6 antibodies or antibody fragments,using standard techniques. (See, e.g., Green et al., “Production ofPolyclonal Antisera,” in Methods In Molecular Biology: ImmunochemicalProtocols 1-12 (Manson, ed., Humana Press 1992). See also Coligan atpages 2.4.1-2.4.7.) Alternatively, monoclonal anti-idiotype antibodiescan be prepared using anti-zB7H6 antibodies or antibody fragments asimmunogens with the techniques, described above. As another alternative,humanized anti-idiotype antibodies or subhuman primate anti-idiotypeantibodies can be prepared using the above-described techniques. Methodsfor producing anti-idiotype antibodies are described, for example, inU.S. Pat. No. 5,208,146 to Irie; U.S. Pat. No. 5,637,677 to Greene, et.al., and Varthakavi and Minocha, J. Gen. Virol. 77:1875, 1996.

An anti-zB7H6 antibody can be conjugated with a detectable label to forman anti-zB7H6 immunoconjugate. Suitable detectable labels include, forexample, a radioisotope, a fluorescent label, a chemiluminescent label,an enzyme label, a bioluminescent label or colloidal gold. Methods ofmaking and detecting such detectably-labeled immunoconjugates arewell-known to those of ordinary skill in the art, and are described inmore detail below.

The detectable label can be a radioisotope that is detected byautoradiography. Isotopes that are particularly useful for the purposeof the present invention are ³H, ¹²⁵I, ¹³¹I, ³⁵S and ¹⁴C.

Anti-zB7H6 immunoconjugates can also be labeled with a fluorescentcompound. The presence of a fluorescently-labeled antibody is determinedby exposing the immunoconjugate to light of the proper wavelength anddetecting the resultant fluorescence. Fluorescent labeling compoundsinclude fluorescein isothiocyanate, rhodamine, phycoerytherin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

Alternatively, anti-zB7H6 immunoconjugates can be detectably labeled bycoupling an antibody component to a chemiluminescent compound. Thepresence of the chemiluminescent-tagged immunoconjugate is determined bydetecting the presence of luminescence that arises during the course ofa chemical reaction. Examples of chemiluminescent labeling compoundsinclude luminol, isoluminol, an aromatic acridinium ester, an imidazole,an acridinium salt and an oxalate ester.

Similarly, a bioluminescent compound can be used to label anti-zB7H6immunoconjugates of the present invention. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Bioluminescent compounds that are useful forlabeling include luciferin, luciferase and aequorin.

Alternatively, anti-zB7H6 immunoconjugates can be detectably labeled bylinking an anti-zB7H6 antibody component to an enzyme. When theanti-zB7H6-enzyme conjugate is incubated in the presence of theappropriate substrate, the enzyme moiety reacts with the substrate toproduce a chemical moiety which can be detected, for example, byspectrophotometric, fluorometric or visual means. Examples of enzymesthat can be used to detectably label polyspecific immunoconjugatesinclude β-galactosidase, glucose oxidase, peroxidase and alkalinephosphatase.

Those of skill in the art will know of other suitable labels which canbe employed in accordance with the present invention. The binding ofmarker moieties to anti-zB7H6 antibodies can be accomplished usingstandard techniques known to the art. Typical methodology in this regardis described by Kennedy et al., Clin. Chim. Acta 70:1, 1976; Schurs etal., Clin. Chim. Acta 81:1, 1977; Shih et al., Int'J. Cancer 46:1101,1990; Stein et al., Cancer Res. 50:1330, 1990; and Coligan, supra.

Moreover, the convenience and versatility of immunochemical detectioncan be enhanced by using anti-zB7H6 antibodies that have been conjugatedwith avidin, streptavidin, and biotin. (See, e.g., Wilchek et al.(eds.), “Avidin-Biotin Technology,” Methods In Enzymology (Vol. 184)(Academic Press 1990); Bayer et al., “Immunochemical Applications ofAvidin-Biotin Technology,” in Methods In Molecular Biology (Vol. 10)149-162 (Manson, ed., The Humana Press, Inc. 1992).)

Methods for performing immunoassays are well-established. (See, e.g.,Cook and Self, “Monoclonal Antibodies in Diagnostic Immunoassays,” inMonoclonal Antibodies: Production, Engineering, and Clinical Application180-208 (Ritter and Ladyman, eds., Cambridge University Press 1995);Perry, “The Role of Monoclonal Antibodies in the Advancement ofImmunoassay Technology,” in Monoclonal Antibodies: Principles andApplications 107-120 (Birch and Lennox, eds., Wiley-Liss, Inc. 1995);Diamandis, Immunoassay (Academic Press, Inc. 1996).)

The present invention also contemplates kits for performing animmunological diagnostic assay for zB7H6 gene expression. Such kitscomprise at least one container comprising an anti-zB7H6 antibody. A kitmay also comprise a second container comprising one or more reagentscapable of indicating the presence of zB7H6 antibody. Examples of suchindicator reagents include detectable labels such as a radioactivelabel, a fluorescent label, a chemiluminescent label, an enzyme label, abioluminescent label, colloidal gold, and the like. A kit may alsocomprise a means for conveying to the user that zB7H6 antibodies areused to detect zB7H6 protein. For example, written instructions maystate that the enclosed antibody or antibody fragment can be used todetect zB7H6. The written material can be applied directly to acontainer, or the written material can be provided in the form of apackaging insert.

V. Anti-zB7H6 Antibody-Drug Conjugates

In certain aspects, the present invention provides an anti-zB7H6antibody-drug conjugate. An “anti-zB7H6 antibody-drug conjugate” as usedherein refers to an anti-zB7H6 antibody (as described in Section IV,supra) conjugated to a therapeutic agent. Such anti-zB7H6 antibody-drugconjugates produce clinically beneficial effects on zB7H6-expressingcells when administered to a subject, such as, for example, a subjectwith a zB7H6-expressing cancer, typically when administered alone butalso in combination with other therapeutic agents.

In typical embodiments, an anti-zB7H6 antibody is conjugated to acytotoxic agent, such that the resulting antibody-drug conjugate exertsa cytotoxic or cytostatic effect on a zB7H6-expressing cell (e.g., azB7H6-expressing cancer cell) when taken up or internalized by the cell.Particularly suitable moieties for conjugation to antibodies arechemotherapeutic agents, prodrug converting enzymes, radioactiveisotopes or compounds, or toxins. For example, an anti-zB7H6 antibodycan be conjugated to a cytotoxic agent such as a chemotherapeutic agent(see infra) or a toxin (e.g., a cytostatic or cytocidal agent such as,for example, abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin).Examples of additional agents that are useful for conjugating to ananti-zB7H6 antibody are provided infra.

In other embodiments, an anti-zB7H6 antibody is conjugated to a pro-drugconverting enzyme. The pro-drug converting enzyme can be recombinantlyfused to the antibody or chemically conjugated thereto using knownmethods. Exemplary pro-drug converting enzymes are carboxypeptidase G2,β-glucuronidase, penicillin-V-amidase, penicillin-G-amidase,β-lactamase, β-glucosidase, nitroreductase and carboxypeptidase A.

Techniques for conjugating therapeutic agents to proteins, and inparticular to antibodies, are well-known. (See, e.g., Amon et al.,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy,”in Monoclonal Antibodies And Cancer Therapy (Reisfeld et al. eds., AlanR. Liss, Inc., 1985); Hellstrom et al., “Antibodies For Drug Delivery,”in Controlled Drug Delivery (Robinson et al. eds., Marcel Deiker, Inc.,2nd ed. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In CancerTherapy: A Review,” in Monoclonal Antibodies '84: Biological AndClinical Applications (Pinchera et al. eds., 1985); “Analysis, Results,and Future Prospective of the Therapeutic Use of Radiolabeled AntibodyIn Cancer Therapy,” in Monoclonal Antibodies For Cancer Detection AndTherapy (Baldwin et al. eds., Academic Press, 1985); and Thorpe et al.,1982, Immunol. Rev. 62:119-58. See also, e.g., PCT publication WO89/12624.)

In certain variations, in accordance with methods described herein, ananti-zB7H6 antibody-drug conjugate is internalized and accumulateswithin a zB7H6-expressing cell, where antibody-drug conjugate exerts atherapeutic effect (e.g., a cytotoxic or cytostatic effect). Methods fordetermining accumulation and rates of accumulation are found in, forexample, WO 2004/010957, entitled “Drug Conjugates and Their Use forTreating Cancer, an Autoimmune Disease or an Infectious Disease.”

In typical embodiments, when using an anti-zB7H6 antibody conjugated toa therapeutic agent (e.g., a drug or a prodrug converting enzyme), theagent is preferentially active when internalized by zB7H6-expressingcells (e.g., cells of a zB7H6-expressing cancer) to be treated. In otherembodiments, the anti-zB7H6 antibody-drug conjugate is not internalized,and the drug is effective to exert a therapeutic effect (e.g., depletionor inhibition of growth of zB7H6-expressing cells) by binding to thecell membrane.

To minimize activity of a therapeutic agent outside a zB7H6-expressingcell (e.g., a zB7H6-expressing cancer cell), a therapeutic agent istypically conjugated in a manner that reduces its activity unless it iscleaved off the antibody (e.g., by hydrolysis or by a cleaving agent).In such embodiments, the therapeutic agent is attached to the antibodywith a cleavable linker that is sensitive to cleavage in theintracellular environment of the zB7H6-expressing cell but is notsubstantially sensitive to the extracellular environment, such that theconjugate is cleaved from the antibody when it is internalized by thezB7H6-expressing cell (e.g., in the endosomal or, for example, by virtueof pH sensitivity or protease sensitivity, in the lysosomal environmentor in a caveolea). (See Section V(A), infra.)

Further, in certain embodiments, an antibody-drug conjugate comprises atherapeutic agent that is charged relative to the plasma membrane,thereby further minimizing the ability of the agent to cross the plasmamembrane once internalized by a cell. As used herein, a “charged agent”means an agent that (a) is polarized, such that one region of the agenthas a charge relative to the plasma membrane, or (b) has a net chargerelative to the plasma membrane.

Typically, an anti-zB7H6 antibody-drug conjugate is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side-effects). In certain specific embodiments, theanti-zB7H6 antibody-drug conjugate is 40% pure, more typically about 50%pure, and most typically about 60% pure. In other specific embodiments,the anti-CD70 ADC or ADC derivative is at least approximately 60-65%,65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-98% pure. Inanother specific embodiment, the anti-CD70 ADC or ADC derivative isapproximately 99% pure.

A. Linkers

Typically, a zB7H6 antibody-drug conjugate comprises a linker regionbetween the therapeutic agent and the anti-zB7H6 antibody. As notedsupra, in certain embodiments, the linker is cleavable underintracellular conditions, such that cleavage of the linker releases thetherapeutic agent from the antibody in the intracellular environment.

For example, in some embodiments, the linker is cleavable by a cleavingagent that is present in the intracellular environment (e.g., within alysosome or endosome or caveolea). The linker can be, e.g., a peptidyllinker that is cleaved by an intracellular peptidase or protease enzyme,including, but not limited to, a lysosomal or endosomal protease.Typically, the peptidyl linker is at least two amino acids long or atleast three amino acids long. Cleaving agents can include cathepsins Band D and plasmin, all of which are known to hydrolyze dipeptide drugderivatives resulting in the release of active drug inside target cells(see, e.g., Dubowchik and Walker, Pharm. Therapeutics 83:67-123, 1999).Most typical are peptidyl linkers that are cleavable by enzymes that arepresent in zB7H6-expressing cells. For example, a peptidyl linker thatis cleavable by the thiol-dependent protease cathepsin-B, which ishighly expressed in cancerous tissue, can be used (e.g., a Phe-Leu or aGly-Phe-Leu-Gly linker). Other such linkers are described, e.g., in U.S.Pat. No. 6,214,345. In specific embodiments, the peptidyl linkercleavable by an intracellular protease is a Val-Cit (valine-citrulline)linker or a Phe-Lys (phenylalanine-lysine) linker (see, e.g., U.S. Pat.No. 6,214,345, which describes the synthesis of doxorubicin with theval-cit linker). One advantage of using intracellular proteolyticrelease of the therapeutic agent is that the agent is typicallyattenuated when conjugated and the serum stabilities of the conjugatesare typically high.

In other embodiments, the cleavable linker is pH-sensitive, i.e.,sensitive to hydrolysis at certain pH values. Typically, a pH-sensitivelinker is hydrolyzable under acidic conditions. For example, anacid-labile linker that is hydrolyzable in the lysosome (e.g., ahydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) can be used. (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, Pharm.Therapeutics 83:67-123, 1999; Neville et al., Biol. Chem.264:14653-14661, 1989.) Such linkers are relatively stable under neutralpH conditions, such as those in the blood, but are unstable at below pH5.5 or 5.0, the approximate pH of the lysosome. In certain embodiments,the hydrolyzable linker is a thioether linker (such as, e.g., athioether attached to the therapeutic agent via an acylhydrazone bond(see, e.g., U.S. Pat. No. 5,622,929)).

In yet other embodiments, the linker is cleavable under reducingconditions (e.g., a disulfide linker). A variety of disulfide linkersare known in the art, including, for example, those that can be formedusing SATA (N-succinimidyl-5-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene),SPDB and SMPT. (See, e.g., Thorpe et al., Cancer Res. 47:5924-5931,1987; Wawrzynczak et al., Immunoconjugates: Antibody Conjugates inRadioimagery and Therapy of Cancer (C. W. Vogel ed., Oxford U. Press,1987. See also U.S. Pat. No. 4,880,935.)

In yet other variations, the linker is a malonate linker (Johnson etal., Anticancer Res. 15:1387-93, 1995), a maleimidobenzoyl linker (Lauet al., Bioorg-Med-Chem. 3:1299-1304, 1995), or a 3′-N-amide analog (Lauet al., Bioorg-Med-Chem. 3:1305-12, 1995).

Typically, the linker is not substantially sensitive to theextracellular environment. As used herein, “not substantially sensitiveto the extracellular environment,” in the context of a linker, meansthat no more than about 20%, typically no more than about 15%, moretypically no more than about 10%, and even more typically no more thanabout 5%, no more than about 3%, or no more than about 1% of thelinkers, in a sample of an antibody-drug conjugate, are cleaved when theantibody-drug conjugate is present in an extracellular environment(e.g., in plasma). Whether a linker is not substantially sensitive tothe extracellular environment can be determined, for example, byincubating independently with plasma both (a) the antibody-drugconjugate (the “antibody-drug conjugate sample”) and (b) an equal molaramount of unconjugated antibody or therapeutic agent (the “controlsample”) for a predetermined time period (e.g., 2, 4, 8, 16, or 24hours) and then comparing the amount of unconjugated antibody ortherapeutic agent present in the antibody-drug conjugate sample withthat present in the control sample, as measured, for example, by highperformance liquid chromatography.

In some variations, the linker promotes cellular internalization. Incertain embodiments, the linker promotes cellular internalization whenconjugated to the therapeutic agent (i.e., in the milieu of thelinker-therapeutic agent moiety of the antibody-drug conjuage). In yetother embodiments, the linker promotes cellular internalization whenconjugated to both the therapeutic agent and the anti-zB7H6 antibody(i.e., in the milieu of the antibody-drug conjugate).

A variety of linkers that can be used with the present compositions andmethods are described in, for example, WO 2004/010957, entitled “DrugConjugates and Their Use for Treating Cancer, an Autoimmune Disease oran Infectious Disease.”

B. Therapeutic Agents

In accordance with the present invention, any agent that exerts atherapeutic effect on a zB7H6-expressing cell can be used as thetherapeutic agent for conjugation to an anti-zB7H6 antibody. In certainembodiments, such as for treatment of a zB7H6-expressing cancer, thetherapeutic agent is a cytotoxic agent.

Useful classes of cytotoxic agents include, for example, antitubulinagents, auristatins, DNA minor groove binders, DNA replicationinhibitors, alkylating agents (e.g., platinum complexes such ascis-platin, mono(platinum), bis(platinum) and tri-nuclear platinumcomplexes and -carboplatin), anthracyclines, antibiotics, antifolates,antimetabolites, chemotherapy sensitizers, duocarmycins, etoposides,fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas,platinols, pre-forming compounds, purine antimetabolites, puromycins,radiation sensitizers, steroids, taxanes, topoisomerase inhibitors,vinca alkaloids, or the like.

Individual cytotoxic agents include, for example, an androgen,anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine, bleomycin,busulfan, buthionine sulfoximine, camptothecin, carboplatin, carmustine(BSNU), CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide,cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine,dactinomycin (formerly actinomycin), daunorubicin, decarbazine,docetaxel, doxorubicin, an estrogen, 5-fluordeoxyuridine,5-fluorouracil, gramicidin D, hydroxyurea, idarubicin, ifosfamide,irinotecan, lomustine (CCNU), mechlorethamine, melphalan,6-mercaptopurine, methotrexate, mithramycin, mitomycin C, mitoxantrone,nitroimidazole, paclitaxel, plicamycin, procarbizine, streptozotocin,tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine,vincristine, vinorelbine, VP-16 and VM-26.

Particularly suitable cytotoxic agents include, for example, dolastatins(e.g., auristatin E, AFP, MMAF, MMAE), DNA minor groove binders (e.g.,enediynes and lexitropsins), duocarmycins, taxanes (e.g., paclitaxel anddocetaxel), puromycins, vinca alkaloids, CC-1065, SN-38, topotecan,morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin,echinomycin, combretastatin, netropsin, epothilone A and B,estramustine, cryptophysins, cemadotin, maytansinoids, discodermolide,eleutherobin, and mitoxantrone.

In certain embodiments, a cytotoxic agent is a conventionalchemotherapeutic such as, for example, doxorubicin, paclitaxel,melphalan, vinca alkaloids, methotrexate, mitomycin C or etoposide. Inaddition, potent agents such as CC-1065 analogues, calicheamicin,maytansine, analogues of dolastatin 10, rhizoxin, and palytoxin can belinked to an anti-zB7H6-expressing antibody.

In specific variations, the cytotoxic or cytostatic agent is auristatinE (also known in the art as dolastatin-10) or a derivative thereof.Typically, the auristatin E derivative is, e.g., an ester formed betweenauristatin E and a keto acid. For example, auristatin E can be reactedwith paraacetyl benzoic acid or benzoylvaleric acid to produce AEB andAEVB, respectively. Other typical auristatin derivatives include AFP(dimethylvaline-valine-dolaisoleuine-dolaproine-phenylalanine-p-phenylenediamine),MMAF (dovaline-valine-dolaisoleunine-dolaproine-phenylalanine), and MAE(monomethyl auristatin E). The synthesis and structure of auristatin Eand its derivatives are described in U.S. Patent Application PublicationNo. 20030083263; International Patent Publication Nos. WO 2002/088172and WO 2004/010957; and U.S. Pat. Nos. 6,884,869; 6,323,315; 6,239,104;6,034,065; 5,780,588; 5,665,860; 5,663,149; 5,635,483; 5,599,902;5,554,725; 5,530,097; 5,521,284; 5,504,191; 5,410,024; 5,138,036;5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,816,444; and 4,486,414.

In other variations, the cytotoxic agent is a DNA minor groove bindingagent. (See, e.g., U.S. Pat. No. 6,130,237.) For example, in certainembodiments, the minor groove binding agent is a CBI compound. In otherembodiments, the minor groove binding agent is an enediyne (e.g.,calicheamicin).

In certain embodiments, an antibody-drug conjugate comprises ananti-tubulin agent. Examples of anti-tubulin agents include, forexample, taxanes (e.g., Taxol® (paclitaxel), Taxotere® (docetaxel)), T67(Tularik), vinca alkyloids (e.g., vincristine, vinblastine, vindesine,and vinorelbine), and dolastatins (e.g., auristatin E, AFP, MMAF, MMAE,AEB, AEVB). Other antitubulin agents include, for example, baccatinderivatives, taxane analogs (e.g., epothilone A and B), nocodazole,colchicine and colcimid, estramustine, cryptophysins, cemadotin,maytansinoids, combretastatins, discodermolide, and eleutherobin. Insome embodiments, the cytotoxic agent is a maytansinoid, another groupof anti-tubulin agents. For example, in specific embodiments, themaytansinoid is maytansine or DM-1 (ImmunoGen, Inc.; see also Chari etal., Cancer Res. 52:127-131, 1992).

In other embodiments, the cytotoxic agent is an antimetabolite. Theantimetabolite can be, for example, a purine antagonist (e.g.,azothioprine or mycophenolate mofetil), a dihydrofolate reductaseinhibitor (e.g., methotrexate), acyclovir, gangcyclovir, zidovudine,vidarabine, ribavarin, azidothymidine, cytidine arabinoside, amantadine,dideoxyuridine, iododeoxyuridine, poscarnet, or trifluridine.

C. Formation of Anti-zB7H6 Antibody-Drug Conjugates

The generation of anti-zB7H6 antibody-drug conjugates can beaccomplished by any technique known to the skilled artisan. Briefly, ananti-zB7H6 antibody-drug conjugate comprises an anti-zB7H6 antibody, adrug, and optionally a linker that joins the drug and the antibody. Anumber of different reactions are available for covalent attachment ofdrugs to antibodies. This is often accomplished by reaction of the aminoacid residues of the antibody molecule, including the amine groups oflysine, the free carboxylic acid groups of glutamic and aspartic acid,the sulfhydryl groups of cysteine, and the various moieties of thearomatic amino acids. One of the most commonly used non-specific methodsof covalent attachment is the carbodiimide reaction to link a carboxy(or amino) group of a compound to amino (or carboxy) groups of theantibody. Additionally, bifunctional agents such as dialdehydes orimidoesters have been used to link the amino group of a compound toamino groups of the antibody molecule. Also available for attachment ofdrugs to antibodies is the Schiff base reaction. This method involvesthe periodate oxidation of a drug that contains glycol or hydroxygroups, thus forming an aldehyde which is then reacted with the antibodymolecule. Attachment occurs via formation of a Schiff base with aminogroups of the antibody molecule. Isothiocyanates can also be used ascoupling agents for covalently attaching drugs to antibodies. Othertechniques are known to the skilled artisan and within the scope of thepresent invention. Non-limiting examples of such techniques aredescribed in, e.g., U.S. Pat. Nos. 5,665,358; 5,643,573; and 5,556,623.

In some embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. Incertain embodiments, reactive groups are used on the drug and/or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drug, is subsequently reacted with theanti-zB7H6 antibody under appropriate conditions.

D. Assays for Cytotoxic or Cytostatic Activities

In certain embodiments, an anti-zB7H6 antibody-drug conjugate comprisesan anti-zB7H6 antibody conjugated to a cytotoxic agent, such that theantibody-drug conjugate exerts a cytotoxic or cytostatic effect on azB7H6-expressing cell (e.g., a zB7H6-expressing cancer cell).zB7H6-expressing cells that can be assayed for a cytotoxic or cytostaticeffect of an anti-zB7H6 antibody-drug conjugate can be culture celllines such as, for example, those listed in Table 5, infra. Once ananti-zB7H6 antibody-drug conjugate is confirmed as exerting a cytotoxicor cytostatic on zB7H6-expressing cells, its therapeutic value can bevalidated in an appropriate animal model. In preferred embodiments, ananti-zB7H6 antibody-drug conjugate comprising a cytotoxic agent is usedto treat a zB7H6-expressing cancer. Exemplary animal models of variouscancers, which may be used to evaluate therapeutic efficacy of anantibody-drug conjugate of the present invention, are described inSection VI(B) and in Examples 21-27, infra.

Methods of determining whether an agent exerts a cytostatic or cytotoxiceffect on a cell are generally known in the art. Illustrative examplesof such methods are described below. Determination of any of theseeffects on zB7H6-expressing cells indicates that an anti-zB7H6antibody-drug conjugate is useful in the treatment or prevention ofdiseases or disorders having a pathology mediated, at least in part, byaberrant growth or activation of zB7H6-expressing cells, such as, forexample, a zB7H6-expressing cancer.

For determining whether an anti-zB7H6 antibody-drug conjugate exerts acytostatic effect on zB7H6-expressing cells, a thymidine incorporationassay may be used. For example, zB7H6-expressing cells, at a density of5,000 cells/well of a 96-well plate, can be cultured for a 72-hourperiod and exposed to 0.5 μCi of ³H-thymidine during the final 8 hoursof the 72-hour period, and the incorporation of ³H-thymidine into cellsof the culture is measured in the presence and absence of theantibody-drug conjugate.

For determining cytotoxicity, necrosis or apoptosis (programmed celldeath) can be measured. Necrosis is typically accompanied by increasedpermeability of the plasma membrane, swelling of the cell, and ruptureof the plasma membrane. Apoptosis is typically characterized by membraneblebbing, condensation of cytoplasm, and the activation of endogenousendonucleases.

Cell viability can be measured by determining in a cell the uptake of adye such as neutral red, trypan blue, or ALAMAR™ blue (see, e.g., Pageet al., Intl. J. of Oncology 3:473-476, 1993). In such an assay, thecells are incubated in media containing the dye, the cells are washed,and the remaining dye, reflecting cellular uptake of the dye, ismeasured spectrophotometrically. The protein-binding dye sulforhodamineB (SRB) can also be used to measure cytoxicity (Skehan et al., J. Nat'lCancer Inst. 82:1107-12, 1990).

Alternatively, a tetrazolium salt, such as MTT, is used in aquantitative calorimetric assay for mammalian cell survival andproliferation by detecting living, but not dead, cells (see, e.g.,Mosmann, J. Immunol. Methods 65:55-63, 1983).

Apoptosis can be quantitated by measuring, for example, DNAfragmentation. Commercial photometric methods for the quantitative invitro determination of DNA fragmentation are available. Examples of suchassays, including TUNEL (which detects incorporation of labelednucleotides in fragmented DNA) and ELISA-based assays, are described inBiochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in acell. For example, as with necrosis, loss of plasma membrane integritycan be determined by measuring uptake of certain dyes (e.g., afluorescent dye such as, for example, acridine orange or ethidiumbromide). A method for measuring apoptotic cell number has previouslybeen described by Duke and Cohen, Current Protocols In Immunology(Coligan et al. eds., 1992, pp. 3.17.1-3.17.16). Cells can be alsolabeled with a DNA dye (e.g., acridine orange, ethidium bromide, orpropidium iodide) and the cells observed for chromatin condensation andmargination along the inner nuclear membrane. Other morphologicalchanges that can be measured to determine apoptosis include, e.g.,cytoplasmic condensation, increased membrane blebbing, and cellularshrinkage.

The presence of apoptotic cells can be measured in both the attached and“floating” compartments of the cultures. For example, both compartmentscan be collected by removing the supernatant, trypsining the attachedcells, combining the preparations following a centrifugation wash step(e.g., 10 minutes, 2000 rpm), and detecting apoptosis (e.g., bymeasuring DNA fragmentation). (See, e.g., Piazza et al., Cancer Research55:3110-16, 1995).

VI. Methods of Use

A. General

In another aspect, the present invention provides methods of modulatingactivity (e.g., cytolytic activity) of an NKp30-expressing cell,including, for example, natural killer (NK) cells and T cells (e.g.,CD8⁺ T cells). Such methods include, e.g., methods for treatment ofdiseases or disorders associated with either increased or decreasedactivity of an NKp30-expressing cell. In some embodiments, the methodsinclude contacting an NKp30-expressing cell with a zB7H6 polypeptide, oran agent capable of mimicking the interaction of zB7H6 with NKp30 (e.g.,a zB7H6 anti-idiotypic antibody), in an amount effective to triggerNKp30-mediated activity (e.g., cytolytic activity). The zB7H6polypeptides can be in either soluble or immobilized (e.g.,cell-membrane-bound) form; for example, in specific variations, a methodof enhancing activity of an NKp30-expressing cell includes contacting anNKp30-expressing cell with an isolated, soluble polypeptide comprising apolypeptide segment that has at least 90% or at least 95% sequenceidentity with the amino acid sequence set forth in residues 25-266 ofSEQ ID NO:2, or contacting an NKp30-expressing cell with a cellexpressing a recombinant, membrane-bound zB7H6 polypeptide. In othervariations, the methods include contacting a cell expressing functionalzB7H6, in the presence of an NKp30-expressing cell, with an effectiveamount of an anti-zB7H6 antibody or other agent capable of interferingwith the interaction of zB7H6 with NKp30. Such methods can be performedin vitro, ex vivo, or in vivo.

In certain preferred variations, methods of modulating NK cell activityare provided, including, for example, methods for treatment of diseasesor disorders associated with either increased or decreased NK cellactivity. In some embodiments, the methods include contacting an NK cellwith a zB7H6 polypeptide, or an agent capable of mimicking theinteraction of zB7H6 with NKp30 (e.g., a zB7H6 anti-idiotypic antibody),in an amount effective to trigger NKp30-mediated NK cell cytolyticactivity. The zB7H6 polypeptides can be in either soluble or immobilized(e.g., cell-membrane-bound) form; for example, in specific variations, amethod of enhancing NK cell activity includes contacting a human NK cellwith an isolated, soluble polypeptide comprising a polypeptide segmentthat has at least 90% or at least 95% sequence identity with the aminoacid sequence set forth in residues 25-266 of SEQ ID NO:2, or contactinga human NK cell with a cell expressing a recombinant, membrane-boundzB7H6 polypeptide. In other variations, the methods include contacting acell expressing functional zB7H6, in the presence of an NK cell, with aneffective amount of an anti-zB7H6 antibody or other agent capable ofinterfering with the interaction of zB7H6 with NKp30. Such methods canbe performed in vitro, ex vivo, or in vivo.

In other embodiments, methods of modulating NKp30-expressing T cellactivity are provided, including, for example, methods for treatment ofdiseases or disorders associated with either increased or decreasedactivity of NKp30-expressing T cells. Certain T cells, including CD8⁺ Tcells, have been shown to express NKp30. (See, e.g., Srivastava andSrivastava, Leuk. Res. 30:37-46, 2006.) Accordingly, in someembodiments, the methods include contacting an NKp30-expressing T cell(e.g., a CD8⁺ T cell) with a zB7H6 polypeptide, or an agent capable ofmimicking the interaction of zB7H6 with NKp30 (e.g., a zB7H6anti-idiotypic antibody), in an amount effective to triggerNKp30-mediated T cell activity (e.g., cytolytic activity). The zB7H6polypeptides can be in either soluble or immobilized (e.g.,cell-membrane-bound) form; for example, in specific variations, a methodof enhancing activity of an NKp30-expressing T cell includes contactingan NKp30-expressing T cell with an isolated, soluble polypeptidecomprising a polypeptide segment that has at least 90% or at least 95%sequence identity with the amino acid sequence set forth in residues25-266 of SEQ ID NO:2, or contacting an NKp30-expressing T cell with acell expressing a recombinant, membrane-bound zB7H6 polypeptide. Inother variations, the methods include contacting a cell expressingfunctional zB7H6, in the presence of an NKp30-expressing T cell, with aneffective amount of an anti-zB7H6 antibody or other agent capable ofinterfering with the interaction of zB7H6 with NKp30. Such methods canbe performed in vitro, ex vivo, or in vivo.

As noted above, in particular variations, the method is a method oftreating a disease or disorder associated with NK cell activity. Forexample, in some embodiments, the method includes administering aneffective amount of a soluble zB7H6 polypeptide, or an agent capable ofmimicking the interaction of zB7H6 with NKp30 (e.g., a zB7H6anti-idiotypic antibody), to a subject suffering from, or at an elevatedrisk of developing, a disease or disorder characterized by insufficientnatural killer (NK) cell activity (e.g., a cancer or an infectiousdisease). In alternative embodiments, the method includes administeringan effective amount of an anti-zB7H6 antibody or other agent capable ofinterfering with the interaction of zB7H6 with NKp30 to a subjectsuffering from, or at an elevated risk of developing, anNK-cell-mediated disease or disorder (for example, NK-cell-mediatedallograft rejection such as, e.g., NK-cell-mediated bone marrow cell(BMC) allograft rejection).

In some variations, a soluble zB7H6 polypeptide is used as animmunostimulatory agent for cancer therapy. A variety of secreted,immunomodulatory proteins are known to stimulate anti-tumor responses inanimal models via stimulation of the immune system (see generallyRosenberg (ed.), Principles and practice of the biologic therapy ofcancer (Lippincott Williams & Wilkins, Philadelphia, Pa., 3rd ed.2000)). For example, the use of IL-2 and IFN-α are used for thetreatments of metastatic melanoma and renal cell carcinoma. (See, e.g.,Atkins et al., J. Clin. Oncol. 17:2105-16, 1999; Fyfe et al., J. Clin.Oncol. 13:688-96, 1995; Jonasch and Haluska, Oncologist 6:34-55, 2001.)The proposed mechanism of action of these cytokines includes enhancementof direct tumor cell killing by CD8⁺ T cells and NK cells. Soluble zB7H6receptors as described herein may be used in a similar manner to enhancedirect tumor killing by NK cells or CD8⁺ T cells via induction ofNKp30-mediated cytolytic activity.

A soluble zB7H6 polypeptide can also be used as an immunostimulatoryagent for the treatment of infectious disease, including, e.g., viralinfections. NK cells constitute the first line of defense againstinvading pathogens, and usually become activated in an early phase ofviral infection. (See, e.g., Ahmad and Alvarez, J. Leukoc. Biol.76:743-759, 2004; Shresta et al., Virology 319:262-273, 2004.) CD8⁺ Tcells have also been shown to play a role in mediating immune responsesto infectious pathogens. (See, e.g., Wong and Palmer, Annu. Rev.Immunol. 21:29-70, 2003.) Current treatments for infectious diseaseinclude immune system stimulants known to promote, inter alia, NK and Tcell activity. Such treatments include, for example, the use of IL-2 asa therapeutic in HIV infection (see, e.g., Smith, AIDS 15 Suppl2:S28-35, 2001), as well as the use of IFN-α in the treatment of HCVinfection (see, e.g., Ahmad and Alvarez, supra). The potential fortreating infectious disease via immunomodulatory proteins that increaseNK cell activity is further underscored by observations that effectivetherapy in HCV-infected individuals correlated to their increase in NKcell activity: in the individuals in whom the therapy failed to increasean NK cell response, no decrease in viremia was observed. (See van Thielet al., Dig. Dis. Sci. 39:970-976, 1994; Wozniakowska-Gesicka et al.,Pol. Merkuriusz Lek. 8:376-377, 2000; Bonavita et al., Int. J. TissueReact. 15:11-16, 1993.) Thus, a soluble zB7H6 polypeptide capable ofstimulating NKp30-mediated NK or CD8⁺ T cell activity may be used topromote NK-mediated or CD8⁺-T-cell-mediated anti-pathogen (e.g.,anti-viral) defense mechanisms to treat infectious disease.

In other variations, an anti-zB7H6 antibody is used to suppressNK-cell-mediated bone marrow allograft rejection. Bone marrowtransplantation (BMT) has become an accepted method of therapy for thetreatment of various hematologic malignancies. The efficacy ofallogeneic BMT is limited, however, by certain obstacles such as, e.g.,rejection of the graft. There is ample evidence that NK cells are abarrier to the engraftment of bone marrow allografts and that they alonecan mediate the specificity of BMC rejection in mice. (See, e.g., Murphyet al., J. Exp. Med. 165:1212-1217, 1987; Murphy et al., J. Exp. Med.166:1499-1509, 1987; Murphy et al., J. Immunol. 144:3305-3311, 1990;Murphy et al., Eur. J. Immunol. 20:1729-1734, 1990; Murphy et al.,Immunol. Rev. 181:279-289, 2001.) Clinically, allograft resistanceobserved in patients with SCID who have received HLA-mismatched BMTsdepleted of T cells, without cytoreductive conditioning, is attributedto high activity of NK cells from the donor. (See O'Reilly et al., Vox.Sang. 51:81-86, 1986.) Accordingly, antibodies against the extracellulardomain of zB7H6 and capable of inhibiting the interaction of zB7H6 withNKp30, as described herein, may be used during BMT to inhibit NK cellcytolytic activity against allografts and thereby treat or prevent BMCallograft rejection.

In yet other embodiments, an anti-zB7H6 antibody is used to induceantibody dependent cellular cyotoxicity (ADCC) or complement dependentcytotoxicity (CDC) against zB7H6-expressing cells such as, for example,zB7H6-expressing cancer cells. Antibody therapy has been particularlysuccessful in cancer treatment because certain tumors either displayunique antigens, lineage-specific antigens, or antigens present inexcess amounts relative to normal cells. Experimental evidencedemonstrates that zB7H6 is, relative to normal tissues, highly expressedby many tumor-derived cell lines, including cell lines derived fromcancers of the colon, liver, cervix, lung, pancreas, and prostate, aswell as those derived from various cancers of the blood such asprohemocytic leukemia, B-cell lymphoma, monocytic lymphoma,erythroleukemia, Burkitt's lymphoma, or chronic myelogenous leukemia.This evidence indicates that zB7H6 is a novel tumor-specific ortumor-associated antigen, and that an anti-zB7H6 antibody may be used asan anti-tumor therapeutic. One of the mechanisms associated with theanti-tumor activity of monoclonal antibody therapy is antibody dependentcellular cytotoxicity (ADCC). In ADCC, monoclonal antibodies bind to atarget cell (e.g., cancer cell) and specific effector cells expressingreceptors for the monoclonal antibody (e.g., NK cells, CD8⁺ T cells,monocytes, granulocytes) bind the monoclonal antibody/target cellcomplex resulting in target cell death.

Accordingly, in some embodiments, an anti-zB7H6 antibody comprising anFc region with effector function is used to induce antibody dependentcellular cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC)against a zB7H6-expressing cell. Methods for inducing ADCC generallyinclude contacting the zB7H6-expressing cell with an effective amount ananti-zB7H6 antibody comprising an Fc region having ADCC activity,wherein the contacting step is in the presence of a cytolytic immuneeffector cell expressing an Fc receptor having cytolytic activity.Immune effector cells expressing cytolytic Fc receptors (e.g., FcγRIIIαor CD16) include, for example, NK cells as well certain CD8⁺ T cells.Methods for inducing CDC generally include contacting thezB7H6-expressing cell with an effective amount an anti-zB7H6 antibodycomprising an Fc region having CDC activity, wherein the contacting stepis in the presence of complement. zB7H6-expressing cells that can betargeted for killing using such methods include, for example, cancercells, such as, e.g., colon cancer cells, liver cancer cells, cervicalcancer cells, lung cancer cells, pancreatic cancer cells, prostatecancer cells, prohemocytic leukemia cells, B-cell lymphoma cells,monocytic lymphoma cells, erythroleukemia cells, Burkitt's lymphomacells, and chronic myelogenous leukemia cells, to name a few.

In related embodiments, an anti-zB7H6 antibody comprising an Fc regionwith effector function is used to treat a zB7H6-expressing cancer in asubject. Such methods generally include administering to a subject aneffective amount of an anti-zB7H6 antibody comprising an Fc regionhaving ADCC activity and/or CDC activity. zB7H6-expressing cancersparticularly amenable to treatment using such methods include, forexample, cancers of the colon, liver, cervix, lung, pancreas, orprostate, as well as cancers of the blood such as, e.g., prohemocyticleukemia, B-cell lymphoma, monocytic lymphoma, erythroleukemia,Burkitt's lymphoma, or chronic myelogenous leukemia.

In yet other embodiments, an anti-zB7H6 antibody-drug conjugate (seeSection V, supra) is used to deliver a therapeutic agent to azB7H6-expressing cell, where the agent exerts a therapeutic effect. Incertain preferred variations utilizing an anti-zB7H6 antibody-drugconjugate, the therapeutic agent is a cytotoxic agent that exerts acytotoxic or cytostatic effect on a zB7H6-expressing cell, such as azB7H6-expressing cancer cell. As indicated above, experimental evidencedemonstrates that zB7H6 is, relative to normal tissues, highly expressedby many tumor-derived cell lines, including cell lines derived fromcancers of the colon, liver, cervix, lung, pancreas, and prostate, aswell as those derived from various cancers of the blood such asprohemocytic leukemia, B-cell lymphoma, monocytic lymphoma,erythroleukemia, Burkitt's lymphoma, or chronic myelogenous leukemia.This evidence indicates that zB7H6 is a novel tumor-specific ortumor-associated antigen useful for targeting agents having therapeuticefficacy in cancer treatment, particularly cytotoxic agents that candeplete or inhibit the growth of tumor cells. Accordingly, in someembodiments, an anti-zB7H6 antibody-drug conjugate, comprising ananti-zB7H6 antibody conjugated to a cytotoxic agent, is used to treat azB7H6-expressing cancer.

In each of the embodiments of the treatment methods described herein,the soluble zB7H6 polypeptide, antibody, or other zB7H6-related agent(including, e.g., a zB7H6 polynucleotide or anti-zB7H6 antibody-drugconjugate) is delivered in a manner consistent with conventionalmethodologies associated with management of the disease or disorder forwhich treatment is sought. In accordance with the disclosure herein, aneffective amount of the agent is administered to a subject in need ofsuch treatment for a time and under conditions sufficient to prevent ortreat the disease or disorder.

Subjects for administration of soluble zB7H6 polypeptides, antibodies,or other zB7H6-related agents as described herein include patients athigh risk for developing a particular disease or disorder associatedwith NK cell activity as well as patients presenting with an existing NKcell-associated disease or disorder. In certain embodiments, the subjecthas been diagnosed as having the disease or disorder for which treatmentis sought. Further, subjects can be monitored during the course oftreatment for any change in the disease or disorder (e.g., for anincrease or decrease in clinical symptoms of the disease or disorder).Also, in some variations, the subject does not suffer from anotherdisease or disorder requiring treatment that involves mimicking orblocking the interaction of zB7H6 with a cognate receptor.

In prophylactic applications, pharmaceutical compositions or medicantsare administered to a patient susceptible to, or otherwise at risk of, aparticular disease in an amount sufficient to eliminate or reduce therisk or delay the outset of the disease. In therapeutic applications,compositions or medicants are administered to a patient suspected of, oralready suffering from such a disease in an amount sufficient to cure,or at least partially arrest, the symptoms of the disease and itscomplications. An amount adequate to accomplish this is referred to as atherapeutically- or pharmaceutically-effective dose or amount. In bothprophylactic and therapeutic regimes, agents are usually administered inseveral dosages until a sufficient response (e.g., triggering ofappropriate NK cell activity or inhibition of inappropriate NK cellactivity) has been achieved. Typically, the response is monitored andrepeated dosages are given if the desired response starts to fade.

To identify subject patients for treatment according to the methods ofthe invention, accepted screening methods may be employed to determinerisk factors associated with specific NK cell-associated disorders or todetermine the status of an existing disorder identified in a subject.Such methods can include, for example, determining whether an individualhas relatives who have been diagnosed with a particular disease.Screening methods can also include, for example, conventional work-upsto determine familial status for a particular disease known to have aheritable component (for example, in the case of BMT, clinical studieshave shown that the presence of certain HLA-C alleles correlates with anincreased risk for BM allograft rejection [see Scott et al., Blood92:486-44871, 1998] and various cancers are also known to have certaininheritable components). Inheritable components of cancers include, forexample, mutations in multiple genes that are transforming (e.g., Ras,Raf, EGFR, cMet and others), the presence or absence of certain HLA andkiller inhibitory receptor (KIR) molecules, or mechanisms by whichcancer cells are able to modulate immune suppression of cells like NKcells and T cells, either directly or indirectly (see, e.g., Ljunggrenand Malmberg, Nature Rev. Immunol. 7:329-339, 2007; Boyton and Altmann,Clin. Exp. Immunol. 149:1-8, 2007). Toward this end, nucleotide probescan be routinely employed to identify individuals carrying geneticmarkers associated with a particular disease of interest. In addition, awide variety of immunological methods are known in the art that areuseful to identify markers for specific diseases. For example, variousELISA immunoassay methods are available and well-known in the art thatemploy monoclonal antibody probes to detect antigens associated withspecific tumors. Screening may be implemented as indicated by knownpatient symptomology, age factors, related risk factors, etc. Thesemethods allow the clinician to routinely select patients in need of themethods described herein for treatment. In accordance with thesemethods, modulation of NK cell activity may be implemented as anindependent treatment program or as a follow-up, adjunct, or coordinatetreatment regimen to other treatments.

For administration, the zB7H6 polypeptide, antibody, or otherzB7H6-related agent is formulated as a pharmaceutical composition. Apharmaceutical composition comprising a soluble zB7H6 polypeptide,anti-zB7H6 antibody, or other agent can be formulated according to knownmethods to prepare pharmaceutically useful compositions, whereby thetherapeutic molecule is combined in a mixture with a pharmaceuticallyacceptable carrier. A composition is said to be a “pharmaceuticallyacceptable carrier” if its administration can be tolerated by arecipient patient. Sterile phosphate-buffered saline is one example of apharmaceutically acceptable carrier. Other suitable carriers arewell-known to those in the art. (See, e.g., Gennaro (ed.), Remington'sPharmaceutical Sciences (Mack Publishing Company, 19th ed. 1995).)Formulations may further include one or more excipients, preservatives,solubilizers, buffering agents, albumin to prevent protein loss on vialsurfaces, etc.

A pharmaceutical composition comprising a zB7H6 polypeptide, antibody,or other zB7H6-related agent is administered to a subject in aneffective amount. According to the methods of the present invention, thepolypeptide, antibody, or other agent may be administered to subjects bya variety of administration modes, including, for example, byintramuscular, subcutaneous, intravenous, intra-atrial, intra-articular,parenteral, intranasal, intrapulmonary, transdermal, intrapleural,intrathecal, and oral routes of administration. For prevention andtreatment purposes, the agent may be administered to a subject in asingle bolus delivery, via continuous delivery (e.g., continuoustransdermal delivery) over an extended time period, or in a repeatedadministration protocol (e.g., on an hourly, daily, or weekly basis).

Determination of effective dosages in this context is typically based onanimal model studies followed up by human clinical trials and is guidedby determining effective dosages and administration protocols thatsignificantly reduce the occurrence or severity of the subject diseaseor disorder in model subjects. Effective doses of the compositions ofthe present invention vary depending upon many different factors,including means of administration, target site, physiological state ofthe patient, whether the patient is human or an animal, othermedications administered, whether treatment is prophylactic ortherapeutic, as well as the specific activity of the composition itselfand its ability to elicit the desired response in the individual.Usually, the patient is a human, but in some diseases, the patient canbe a nonhuman mammal. Typically, dosage regimens are adjusted to providean optimum therapeutic response, i.e., to optimize safety and efficacy.Accordingly, a therapeutically or prophylactically effective amount isalso one in which any undesired collateral effects are outweighed bybeneficial effects of modulating NKp30-mediated NK cell activity. Foradministration of a soluble zB7H6 polypeptide or an antibody, a dosagetypically ranges from about 0.1 μg to 100 mg/kg or 1 μg/kg to about 50mg/kg, and more usually 10 μg to 5 mg/kg of the subject's body weight.In more specific embodiments, an effective amount of the agent isbetween about 1 μg/kg and about 20 mg/kg, between about 10 μg/kg andabout 10 mg/kg, or between about 0.1 mg/kg and about 5 mg/kg. Dosageswithin this range can be achieved by single or multiple administrations,including, e.g., multiple administrations per day or daily, weekly,bi-weekly, or monthly administrations. For example, in certainvariations, a regimen consists of an initial administration followed bymultiple, subsequent administrations at weekly or bi-weekly intervals.Another regimen consists of an initial administration followed bymultiple, subsequent administrations at monthly or bimonthly intervals.Alternatively, administrations can be on an irregular basis as indicatedby monitoring of NK cell activity and/or clinical symptoms of thedisease or disorder.

Dosage of the pharmaceutical composition may be varied by the attendingclinician to maintain a desired concentration at a target site. Forexample, if an intravenous mode of delivery is selected, localconcentration of the agent in the bloodstream at the target tissue maybe between about 1-50 nanomoles of the composition per liter, sometimesbetween about 1.0 nanomole per liter and 10, 15, or 25 nanomoles perliter depending on the subject's status and projected measured response.Higher or lower concentrations may be selected based on the mode ofdelivery, e.g., trans-epidermal delivery versus delivery to a mucosalsurface. Dosage should also be adjusted based on the release rate of theadministered formulation, e.g., nasal spray versus powder, sustainedrelease oral or injected particles, transdermal formulations, etc. Toachieve the same serum concentration level, for example, slow-releaseparticles with a release rate of 5 nanomolar (under standard conditions)would be administered at about twice the dosage of particles with arelease rate of 10 nanomolar.

A pharmaceutical composition comprising a soluble zB7H6 polypeptide,antibody, or other zB7H6-related composition can be furnished in liquidform, in an aerosol, or in solid form. Liquid forms, are illustrated byinjectable solutions, aerosols, droplets, topological solutions and oralsuspensions. Exemplary solid forms include capsules, tablets, andcontrolled-release forms. The latter form is illustrated by miniosmoticpumps and implants. (See, e.g., Bremer et al., Pharm. Biotechnol.10:239, 1997; Ranade, “Implants in Drug Delivery,” in Drug DeliverySystems 95-123 (Ranade and Hollinger, eds., CRC Press 1995); Bremer etal., “Protein Delivery with Infusion Pumps,” in Protein Delivery:Physical Systems 239-254 (Sanders and Hendren, eds., Plenum Press 1997);Yewey et al., “Delivery of Proteins from a Controlled Release InjectableImplant,” in Protein Delivery: Physical Systems 93-117 (Sanders andHendren, eds., Plenum Press 1997).) Other solid forms include creams,pastes, other topological applications, and the like.

Liposomes provide one means to deliver therapeutic polypeptides to asubject, e.g., intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments.(See, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):S61, 1993; Kim, Drugs 46:618, 1993; Ranade,“Site-Specific Drug Delivery Using Liposomes as Carriers,” in DrugDelivery Systems 3-24 (Ranade and Hollinger, eds., CRC Press 1995).)Liposomes are similar in composition to cellular membranes and as aresult, liposomes can be administered safely and are biodegradable.Depending on the method of preparation, liposomes may be unilamellar ormultilamellar, and liposomes can vary in size with diameters rangingfrom 0.02 μm to greater than 10 μm. A variety of agents can beencapsulated in liposomes: hydrophobic agents partition in the bilayersand hydrophilic agents partition within the inner aqueous space(s).(See, e.g., Machy et al., Liposomes In Cell Biology And Pharmacology(John Libbey 1987); Ostro et al., American J. Hosp. Pharm. 46:1576,1989.) Moreover, it is possible to control the therapeutic availabilityof the encapsulated agent by varying liposome size, the number ofbilayers, lipid composition, as well as the charge and surfacecharacteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (see Scherphof et al., Ann. N.Y. Acad. Sci. 446:368,1985). After intravenous administration, small liposomes (0.1 to 1.0 μm)are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (see Claassenet al., Biochim. Biophys. Acta 802:428, 1984). In addition,incorporation of glycolipid- or polyethelene glycol-derivatizedphospholipids into liposome membranes has been shown to result in asignificantly reduced uptake by the reticuloendothelial system (seeAllen et al., Biochim. Biophys. Acta 1068:133, 1991; Allen et al.,Biochim. Biophys. Acta 1150:9, 1993).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors orcounter-receptors into the liposomes. For example, liposomes, preparedwith a high content of a nonionic surfactant, have been used to targetthe liver. (See, e.g., Japanese Patent 04-244,018 to Hayakawa et al.;Kato et al., Biol. Pharm. Bull. 16:960, 1993.) These formulations wereprepared by mixing soybean phospatidylcholine, α-tocopherol, andethoxylated hydrogenated castor oil (HCO-60) in methanol, concentratingthe mixture under vacuum, and then reconstituting the mixture withwater. A liposomal formulation of dipalmitoylphosphatidylcholine (DPPC)with a soybean-derived sterylglucoside mixture (SG) and cholesterol (Ch)has also been shown to target the liver. (See Shimizu et al., Biol.Pharm. Bull. 20:881, 1997.)

Alternatively, various targeting counter-receptors can be bound to thesurface of the liposome, such as antibodies, antibody fragments,carbohydrates, vitamins, and transport proteins. For example, fortargeting to the liver, liposomes can be modified with branched typegalactosyllipid derivatives to target asialoglycoprotein (galactose)receptors, which are exclusively expressed on the surface of livercells. (See Kato and Sugiyama, Crit. Rev. Ther. Drug Carrier Syst.14:287, 1997; Murahashi et al., Biol. Pharm. Bull. 20:259, 1997.) In amore general approach to tissue targeting, target cells are prelabeledwith biotinylated antibodies specific for a counter-receptor expressedby the target cell. (See Harasym et al., Adv. Drug Deliv. Rev. 32:99,1998.) After plasma elimination of free antibody,streptavidin-conjugated liposomes are administered. In another approach,targeting antibodies are directly attached to liposomes. (See Harasym etal., supra.)

Polypeptides and antibodies can be encapsulated within liposomes usingstandard techniques of protein microencapsulation. (See, e.g., Andersonet al., Infect. Immun. 31:1099, 1981; Anderson et al., Cancer Res.50:1853, 1990; Cohen et al., Biochim. Biophys. Acta 1063:95, 1991;Alving et al. “Preparation and Use of Liposomes in ImmunologicalStudies,” in Liposome Technology (Vol. III) 317 (Gregoriadis, ed., CRCPress, 2nd ed. 1993); Wassef et al., Meth. Enzymol. 149:124, 1987.) Asnoted above, therapeutically useful liposomes may contain a variety ofcomponents. For example, liposomes may comprise lipid derivatives ofpoly(ethylene glycol). (See Allen et al., Biochim. Biophys. Acta 1150:9,1993.)

Degradable polymer micro spheres have been designed to maintain highsystemic levels of therapeutic proteins. Micro spheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer. (See, e.g.,Gombotz and Pettit, Bioconjugate Chem. 6:332, 1995; Ranade, “Role ofPolymers in Drug Delivery,” in Drug Delivery Systems 51-93 (Ranade andHollinger, eds., CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems 45-92 (Sanders and Hendren, eds., PlenumPress 1997); Bartus et al., Science 281:1161, 1998; Putney and Burke,Nature Biotechnology 16:153, 1998; Putney, Curr. Opin. Chem. Biol.2:548, 1998.) Polyethylene glycol (PEG)-coated nanospheres can alsoprovide carriers for intravenous administration of therapeutic proteins.(See, e.g., Gref et al., Pharm. Biotechnol. 10:167, 1997.)

Other dosage forms can be devised by those skilled in the art, as shownby, e.g., Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems (Lea & Febiger, 5th ed. 1990); Gennaro (ed.),Remington's Pharmaceutical Sciences (Mack Publishing Company, 19th ed.1995), and Ranade and Hollinger, Drug Delivery Systems (CRC Press 1996).

zB7H6 polypeptides can be used in the context of gene therapy. Genetherapy can be broadly defined as the transfer of genetic material intoa cell to transiently or permanently alter the cellular phenotype.Numerous methods are being developed for delivery of cytokines, tumorantigens, and additional co-stimulatory molecules via gene therapy tospecific locations within tumor patients (see generally Rosenberg (ed.),Principles and practice of the biologic therapy of cancer (LippincottWilliams & Wilkins, Philadelphia, Pa., 3rd ed. 2000)). Thesemethodologies could be adapted to use zB7H6 DNA or RNA.

Accordingly, in some embodiments, NK cell responses in a subject aremodulated by administration of a nucleic acid encoding a zB7H6 protein,including, e.g., a soluble zB7H6 polypeptide as described herein. Usingsuch zB7H6-encoding nucleic acids, disease or disorders characterized byinsufficient NK cell activity can be treated as generally discussedabove. In the case of nucleic acid therapy, a zB7H6 polypeptide may beexpressed as a soluble receptor, which is secreted from cells to induceNKp30-mediated effects in a manner similar to a soluble zB7H6polypeptide that is directly administered to a subject as describedabove. Alternatively, a zB7H6 polypeptide may be expressed in a formthat maintains association with the surface of the cell in which theprotein is expressed (e.g., with a functional transmembrane domain or aGPI linkage); such embodiments are particularly useful for facilitatingtargeting to particular cells or tissues to maintain localizedNKp30-mediated effects.

zB7H6 polypeptide-encoding nucleic acids for use in therapeutic methodscan be DNA or RNA. A nucleic acid segment encoding the zB7H6 polypeptideis typically linked to regulatory elements, such as a promoter andenhancer, that allow expression of the DNA segment in the intendedtarget cells of a patient. For expression in blood cells, as isdesirable for induction of a NK cell-mediated responses via expressionof zB7H6 polypeptides, promoter and enhancer elements from light orheavy chain immunoglobulin genes or the CMV major intermediate earlypromoter and enhancer are suitable to direct expression. The linkedregulatory elements and coding sequences are often cloned into a vector.

A number of viral vector systems are available including retroviralsystems (see, e.g., Lawrie and Tumin, Cur. Opin. Genet. Develop. 3,102-109, 1993); adenoviral vectors (see, e.g., Bett et al., J. Virol.67, 5911, 1993); adeno-associated virus vectors (see, e.g., Zhou et al.,J. Exp. Med. 179, 1867, 1994), viral vectors from the pox familyincluding vaccinia virus and the avian pox viruses, viral vectors fromthe alpha virus genus such as those derived from Sindbis and SemlikiForest Viruses (see, e.g., Dubensky et al., J. Virol. 70, 508-519,1996), and papillomaviruses (Ohe et al., Human Gene Therapy 6, 325-333,1995; WO 94/12629 (Woo et al.); Xiao & Brandsma, Nucleic Acids. Res. 24,2630-2622, 1996).

Nucleic acids may be also used to decrease the level of functional zB7H6expression in cells. For example, nucleic acids for use in therapeuticmethods may include, for example, inhibitory polynucleotides (e.g.,antisense polynucleotides, small inhibitory RNAs (siRNA), ribozymes, andexternal guide sequences), as well as nucleic acids encoding zB7H6dominant negative variants. Such nucleic acids can be used to inhibitzB7H6 activity in a subject by reducing the level of NKp30 interactionwith functional zB7H6.

DNA encoding a zB7H6 polypeptide, or a vector containing the same, canbe packaged into liposomes. Suitable lipids and related analogs aredescribed by U.S. Pat. Nos. 5,208,036, 5,264,618, 5,279,833 and5,283,185. Vectors and DNA encoding a zB7H6 polypeptide can also beadsorbed to or associated with particulate carriers, examples of whichinclude polymethyl methacrylate polymers and polylactides andpoly(lactide-co-glycolides) (see, e.g., McGee et al., J. Micro Encap.,1996).

Gene therapy vectors or naked DNA can be delivered in vivo byadministration to an individual patient, typically by systemicadministration (e.g., intravenous, intraperitoneal, nasal, gastric,intradermal, intramuscular, subdermal, or intracranial infusion) ortopical application (see e.g., U.S. Pat. No. 5,399,346). DNA can also beadministered using a gene gun. (See Xiao & Brandsma, supra.) The DNAencoding a polypeptide is precipitated onto the surface of microscopicmetal beads. The microprojectiles are accelerated with a shock wave orexpanding helium gas, and penetrate tissues to a depth of several celllayers. For example, The Accel™ Gene Delivery Device manufactured byAgacetus, Inc. Middleton Wis. is suitable. Alternatively, naked DNA canpass through skin into the blood stream simply by spotting the DNA ontoskin with chemical or mechanical irritation (see, e.g., WO 95/05853).

In a further variation, vectors encoding a zB7H6 polypeptide can bedelivered to cells ex vivo, such as cells explanted from an individualpatient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) oruniversal donor hematopoietic stem cells, followed by reimplantation ofthe cells into a patient, usually after selection for cells which haveincorporated the vector.

In certain embodiments, the methods may additionally involve the use ofviruses or other delivery vehicles that specifically recognize a targetcell or tissue (e.g., tumor-targeted viruses, or other delivery vehiclesthat specifically recognize tumor cells.)

Pharmaceutical compositions as described herein may also be used in thecontext of combination therapy. The term “combination therapy” is usedherein to denote that a subject is administered at least onetherapeutically effective dose of a zB7H6-related composition andanother agent. The zB7H6-related composition may be, e.g., a solublezB7H6 polypeptide, an anti-zB7H6 antibody (including, e.g., ananti-zB7H6 antibody-drug conjugate), a zB7H6 mimicking agent such as azB7H6 anti-idiotypic antibody, a zB7H6-encoding polynucleotide, aninhibitory polynucleotide, or other agent that demonstrates zB7H6biological activity, inhibition of zB7H6 biological activity, orspecific binding to zB7H6 (such as, e.g., in the context of targetingtherapeutic agents to zB7H6-expressing cells).

For example, in the context of cancer immunotherapy, compositions havingzB7H6 biological activity can be used as an immunostimulatory agent incombination with chemotherapy, radiation, and myeloablation. zB7H6polypeptides and other agents having zB7H6 biological activity can workin synergy with conventional types of chemotherapy or radiation. Forinstance, in preclinical models of lymphoma and renal cell carcinoma,the combination of IL-2 with doxorubicin (Ehrke et al., Cancer Immunol.Immunother. 42:221-30, 1996), or the combinations of IL-2 (Younes etal., Cell Immunol. 165:243-51, 1995) or IFN-α (Nishisaka et al.,Cytokines Cell Mol. Ther. 6:199-206, 2000) with radiation providedsuperior results over the use of single agents. In this setting, zB7H6polypeptides and zB7H6 mimicking agents can further reduce tumor burdenand allow more efficient killing by the chemotherapeutic. Additionally,lethal doses of chemotherapy or radiation followed by bone marrowtransplantation or stem cell reconstitution could reduce tumor burden toa sufficiently small level (e.g., minimal residual disease) to betterallow for a zB7H6-mediated anti-tumor effect. Examples of this type oftreatment regimen include the uses of IL-2 and IFN-α to modifyanti-cancer responses following myeloablation and transplantation(Porrata et al., Bone Marrow Transplant. 28:673-80, 2001; Slavin andNagler, Cancer J. Sci. Am. Suppl 1:S59-67, 1997; Fefer et al., Cancer J.Sci. Am. Suppl 1:S48-53, 1997). In the case of lymphoma and othercancers, depending on when a zB7H6-related composition is used relativeto the chemotherapeutic agent(s), the zB7H6-related composition may beemployed to directly synergize with the chemotherapeutic agent's effecton the tumor cells or alternatively employed after the chemotherapy tostimulate the immune system. Those skilled in the art would design aprotocol to take advantage of both possibilities.

Compositions of the present invention demonstrating zB7H6 biologicalactivity can be used in combination with other immunomodulatorycompounds including various cytokines and co-stimulatory/inhibitorymolecules. For example, the NK cell stimulatory activity of zB7H6 inmediating an anti-cancer response can be enhanced in patients whencompositions having zB7H6 activity are used with other classes ofimmunomodulatory molecules. These could include, but are not limited to,the use of additional cytokines. For instance, the combined use of IL-2and IL-12 shows beneficial effects in T-cell lymphoma, squamous cellcarcinoma, and lung cancer. (See Zaki et al., J. Invest. Dermatol.118:366-71, 2002; Li et al., Arch. Otolayngol. Head Neck Surg.127:1319-24, 2001; Hiraki et al., Lung Cancer 35:329-33, 2002.) Inaddition, compositions having zB7H6 activity could be combined withreagents that co-stimulate various cell surface molecules found onimmune-based effector cells, such as the activation of CD137. (SeeWilcox et al., J. Clin. Invest. 109:651-9, 2002) or inhibition of CTLA4(Chambers et al., Ann. Rev. Immunol. 19:565-94, 2001.) Alternatively,composition having zB7H6 activity could be used with reagents thatinduce tumor cell apoptosis by interacting with TRAIL-related receptors.(See, e.g., Takeda et al., J. Exp. Med. 195:161-9, 2002; Srivastava,Neoplasia 3:535-46, 2001.) Such reagents include TRAIL ligand, TRAILligand-Ig fusions, anti-TRAIL antibodies, and the like.

In other variations, compositions having zB7H6 activity are used incombination with monoclonal antibody therapy. Such combination therapyis particularly useful for treatment of cancer, in which the use ofmonoclonal antibodies is becoming a standard practice for many tumorsincluding Non-Hodgkins lymphoma (rituximab or RITUXAN®), forms ofleukemia (gemtuzumab or MYLOTARG®), breast cell carcinoma (trastuzumabor HERCEPTIN®), and colon carcinoma (cetuximab or ERBITUX®). Onemechanism by which antibodies mediate an anti-cancer effect is through aprocess referred to as antibody-dependent cell-mediated cytotoxicity(ADCC) in which immune-based cells, including NK cells, macrophages, andneutrophils, kill those cells that are bound by the antibody complex.Accordingly, due to its immunomodulatory in triggering NKp30-mediated NKcell activity, zB7H6 can be used to enhance the effectiveness ofantibody therapy. Examples of this type of treatment paradigm includethe combination use of RITUXAN™ (rituximab) and either IL-2, IL-12, orIFN-α for the treatment of Hodgkin's and Non-Hodgkin's lymphoma. (SeeKeilholz et al., Leuk. Lymphoma 35:641-2, 1999; Ansell et al., Blood99:67-74, 2002; Carson et al., Eur. J. Immunol. 31:3016-25, 2001; Sacchiet al., Haematologica 86:951-8, 2001.)

Pharmaceutical compositions may be supplied as a kit comprising acontainer that comprises a therapeutic polypeptide or polynucleotide asdescribed herein. A therapeutic molecule can be provided, for example,in the form of an injectable solution for single or multiple doses, oras a sterile powder that will be reconstituted before injection.Alternatively, such a kit can include a dry-powder disperser, liquidaerosol generator, or nebulizer for administration of a therapeuticpolypeptide or polynucleotide. Such a kit may further comprise writteninformation on indications and usage of the pharmaceutical composition.For example, such information may include a statement that a zB7H6composition is contraindicated in patients with known hypersensitivityto zB7H6.

B. Cancer Treatment

1. Types of Cancer

As described herein, zB7H6 is an activating ligand for the stimulatoryNK cell receptor, NKp30. As such, in certain variations, agents havingagonistic zB7H6 activity against NKp30 may be used as immunostimulatoryagents for cancer therapy by enhancing direct tumor killing by NK cellsvia induction of NKp30-mediated NK cell cytolytic activity. In addition,as shown by studies described herein, zB7H6 is expressed on a variety oftumor-derived cells. Accordingly, in other variations, a zB7H6 antibodymay be used to direct killing of a zB7H6-expressing cell by activatingthe ADCC or CDC pathway through binding of Fc to Fc receptors and thecomplement protein, C1q. In yet other variations, an anti-zB7H6antibody-drug conjugate, comprising a cytotoxic agent conjugated to ananti-zB7H6 antibody, may be used to deliver a cytotoxic agent tozB7H6-expressing cancer cells, where the cytotoxic agent exerts atherapeutic effect by depleting or inhibition the growth of the cancercells.

Table 4 below lists some cancers amenable to treatment in accordancewith the present invention, organized predominantly by target tissues.

TABLE 4 List of Exemplary Cancer Types  1. Head and Neck cancer a. Brainb. Oral cavity c. Orophyarynx d. Nasopharynx e. Hypopharynx f. Nasalcavities and paranasal sinuses g. Larynx h. Lip  2. Lung cancers a.Non-small cell carcinoma b. Small cell carcinoma  3. GastrointestinalTract cancers a. Colorectal cancer b. Gastric cancer c. Esophagealcancer d. Anal cancer e. Extrahepatic Bile Duct cancer f. Cancer of theAmpulla of Vater g. Gastrointestinal Stromal Tumor (GIST)  4. Livercancer a. Liver Cell Adenoma b. Hepatocellular Carcinoma  5. Breastcancer  6. Gynecologic cancer a. Cervical cancer b. Ovarian cancer c.Vaginal cancer d. Vulvar cancer e. Gestational Trophoblastic Neoplasiaf. Uterine cancer  7. Urinary Tract cancer a. Renal cancer carcinoma b.Prostate cancer c. Urinary Bladder cancer d. Penile cancer e. Urethralcancer  8. Urinary Bladder cancer  9. Neurological Tumors a. Astrocytomaand glioblastoma b. Primary CNS lymphoma c. Medulloblastoma d. Germ Celltumors e. Retinoblastoma 10. Endocrine Neoplasms a. Thyroid cancer b.Pancreatic cancer 1) Islet Cell tumors a) Insulinomas b) Glucagonomas c.Pheochromocytoma d. Adrenal carcinoma e. Carcinoid tumors f. Parathyroidcancinoma g. Pineal gland neoplasms 11. Skin cancers a. Malignantmelanoma b. Squamous Cell carcinoma c. Basal Cell carcinoma d. Kaposi'sSarcoma 12. Bone cancers a. Osteoblastoma b. Osteochondroma c.Osteosarcoma 13. Connective Tissue neoplasms a. Chondroblastoma b.Chondroma 14. Hematopoietic malignancies a. Non-Hodgkin Lymphoma 1)B-cell lymphoma 2) T-cell lymphoma 3) Undifferentiated lymphoma b.Leukemias 1) Chronic Myelogenous Leukemia 2) Hairy Cell Leukemia 3)Chronic Lymphocytic Leukemia 4) Chronic Myelomonocytic Leukemia 5) AcuteMyelocytic Leukemia 6) Acute Lymphoblastic Leukemia c.Myeloproliferative Disorders 1) Multiple Myeloma 2) EssentialThrombocythemia 3) Myelofibrosis with Myeloid Metaplasia 4)Hypereosinophilic Syndrome 5) Chronic Eosinophilic Leukemia 6)Polycythemia Vera d. Hodgkin Lymphoma 15. Childhood Cancers a. Leukemiaand Lymphomas b. Brain cancers c. Neuroblastoma d. Wilm's Tumor(nephroblastoma) e. Phabdomyosarcoma f. Retinoblastoma 16.Immunotherapeutically sensitive cancers a. melanoma b. kidney cancer c.leukemias, lymphomas and myelomas d. breast cancer e. prostate cancer f.colorectal cancer g. cervical cancer h. ovarian cancer i. lung cancer

Some of the cancers listed above, including some of the relevant animalmodels for evaluating the effects of an zB7H6-related agent inaccordance with the present invention on tumor responses, are discussedin further detail below.

a. Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a rare type of cancer affecting mostlyadults. It is a cancer of granulocytes (one of the main types of whiteblood cells). In CML many granulocytes are produced and they arereleased into the blood when they are immature and unable to workproperly. The immature white blood cells are known as blasts. Theproduction of other types of blood cells is also disrupted. Normally,white blood cells repair and reproduce themselves in an orderly andcontrolled manner, but in chronic myeloid leukaemia the process gets outof control and the cells continue to divide and mature abnormally. Thedisease usually develops very slowly, which is why it is called“chronic” myeloid leukemia.

Because CML develops (progresses) slowly, it is difficult to detect inits early stages. Sometimes it is discovered only when a blood test isdone for another reason. The symptoms of CML are often vague andnon-specific and are caused by the increased number of abnormal whiteblood cells in the bone marrow and the reduced number of normal bloodcells: a feeling of fullness or a tender lump on the left side of theabdomen. This is because, in CML, the spleen can become enlarged. Thespleen is an organ which lies just below the ribs on the left side ofthe abdomen. It filters the blood and removes worn-out red blood cells.The swelling of the spleen may also cause pressure on the stomach, whichcan lead to indigestion and poor appetite some people feel tired andlook pale, due to a lack of red blood cells (anemia) due to a lowernumber of platelets in the blood some people may notice that they bleedor bruise more easily. As well as bruising more easily than normal, aspecial type of bruising can be seen. This consists of small blood-likespots usually seen on the legs or in the mouth and is called petechiae.Women may find that their periods become very much heavier. However,these symptoms and signs are rare some people may notice a generaliseditching. Chronic myeloid leukaemia can occur at any age, but it morecommonly affects middle-aged and older people. It is rare in children(cancerbacup internet website). The effects of an agent (e.g., ananti-zB7H6 antibody-drug conjugate) on tumor response can be evaluated,for example, in a human tumor xenograft model, using human CML cellsengrafted in immunodeficient mice (see, e.g., Ren, Leukemia and Lymphoma8:1549-1561, 2002; Van Etten, Blood Cells Mol. Dis. 27:201-205, 2001;Wong and Witte, Oncogene 20:5644-5659, 2001).

b. Multiple Myeloma

Multiple myeloma is a type of cancer affecting certain white blood cellscalled plasma cells. When cancer involves plasma cells, the body keepsproducing more and more of these cells. The unneeded plasma cells—allabnormal and all exactly alike—are called myeloma cells. Myeloma cellstend to collect in the bone marrow and in the hard, outer part of bones.Sometimes they collect in only one bone and form a single mass, ortumor, called a plasmacytoma. In most cases, however, the myeloma cellscollect in many bones, often forming many tumors and causing otherproblems. When this happens, the disease is called multiple myeloma.

Because people with multiple myeloma have an abnormally large number ofidentical plasma cells, they also have too much of one type of antibody.These myeloma cells and antibodies can cause a number of serious medicalproblems. (1) As myeloma cells increase in number, they damage andweaken bones, causing pain and sometimes fractures. Bone pain can makeit difficult for patients to move. (2) When bones are damaged, calciumis released into the blood. This may lead to hypercalcemia—too muchcalcium in the blood. Hypercalcemia can cause loss of appetite, nausea,thirst, fatigue, muscle weakness, restlessness, and confusion. (3)Myeloma cells prevent the bone marrow from forming normal plasma cellsand other white blood cells that are important to the immune system.Patients may not be able to fight infection and disease. (4) The cancercells also may prevent the growth of new red blood cells, causinganemia. Patients with anemia may feel unusually tired or weak. And (5)Multiple myeloma patients may have serious problems with their kidneys.Excess antibody proteins and calcium can prevent the kidneys fromfiltering and cleaning the blood properly. Symptoms of multiple myelomadepend on how advanced the disease is. In the earliest stage of thedisease, there may be no symptoms. When symptoms do occur, patientscommonly have bone pain, often in the back or ribs. Patients also mayhave broken bones, weakness, fatigue, weight loss, or repeatedinfections. When the disease is advanced, symptoms may include nausea,vomiting, constipation, problems with urination, and weakness ornumbness in the legs (National Cancer Institute's Internet website). Theeffects of an agent (e.g., an anti-zB7H6 antibody-drug conjugate) ontumor response can be evaluated in a human tumor xenograft model inimmunodeficient mice, such as described in Miyakawa et al., Biochem.Biophys. Res. Comm n. 313:258-62, 2004.

c. Non-Hodgkin's Lymphoma

Non-Hodgkin's lymphomas are a type of cancer of the lymphatic system.There are two main types of lymphoma. One is called Hodgkin's disease(named after Dr. Hodgkin, who first described it). The other is callednon-Hodgkin's lymphoma. There are about 20 different types ofnon-Hodgkin's lymphoma. In most cases of Hodgkin's disease, a particularcell known as the Reed-Sternberg cell is found in the biopsies. Thiscell is not usually found in other lymphomas, so they are callednon-Hodgkin's lymphoma. This may not seem a very big difference, but itis important because the treatment for Hodgkin's and non-Hodgkin'slymphomas can be very different.

Often, the first sign of a non-Hodgkin's lymphoma is a painless swellingof a lymph node in the neck, armpit or groin. Other symptoms may includeany of the following: night sweats or unexplained high temperatures(fever); loss of appetite, unexplained weight loss and excessivetiredness; children may develop a cough or breathlessness. They may alsocomplain of abdominal pain, or you may notice a lump in your child'sabdomen or persistent itching of the skin all over the body (cancerbacupinternet website). The effects of an agent (e.g., an anti-zB7H6antibody-drug conjugate) on tumor response can be evaluated in anon-Hodgkin's lymphoma xenograft model similar to that described inAnsell et al., Leukemia 18:616-23, 2004.

The classification of Non-Hodgkin's lymphomas most commonly used is theREAL classification system (Ottensmeier, Chemico-Biological Interactions135-136:653-664, 2001.) Specific immunological markers have beenidentified for classifications of lymphomas. For example, follicularlymphoma markers include CD20+, CD3−, CD10+, CD5−; small lymphocyticlymphoma markers include CD20+, CD3−, CD10−, CD5+, CD23+; marginal zoneB cell lymphoma markers include CD20+, CD3−, CD10−, CD23−; diffuse largeB cell lymphoma markers include CD20+, CD3−; mantle cell lymphomamarkers include CD20+, CD3−, CD10−, CD5+, CD23+; peripheral T celllymphoma markers include CD20−, CD3+; primary mediastinal large B celllymphoma markers include CD20+, CD3−, lymphoblastic lymphoma markersinclude CD20−, CD3+, Tdt+, and Burkitt's lymphoma markers include CD20+,CD3−, CD10+, CD5− (Decision Resourses, Non-Hodgkins Lymphoma, Waltham,Mass., February 2002).

Clinical classification of Non Hodgkins lymphoma (NHL) by theInternational Working Formulation breaks down disease into subtypes: (1)low grade (indolent) disease which includes small lymphocytic,consistent with chronic lymphocytic leukemia (SC); follicular,predominantly small cleaved cell (FSC); follicular, mixed small cleavedand large cell (FM); (2) intermediate grade disease which includesfollicular, predominantly large cell (FL); diffuse, small cleaved cell(DSC); diffuse mixed, small and large cell (DM); diffuse, large cleavedor noncleaved cell (DL); and (3) high grade disease which includesimmunoblastic, large cell (IBL); lymphoblastic, convoluted ornonconvoluted cell (LL); and small noncleaved cell, Burkitt's ornon-Burkitts (SNC; (The Non-Hodgkin's Lymphoma Pathologic ClassificationProject, Cancer 49:2112-35, 1982). The Ann Arbor Staging system iscommonly used to stage patients with NHL. Stage I means involvement of asingle lymph node region or localized involvement of a singleextralymphatic organ or site. Stage II means involvement of two or morelymph node regions on the same side of the diaphragm or localizedinvolvement of an extranoldal site or organ and one or more lymph noderegions on the same side of the diaphragm. Stage III means involvementof lymph node regions on both sides of the diaphragm, possiblyaccompanied by localized involvement of an extranodal organ or site.Stage 1V means diffuse or disseminated involvement of one or moredistant extranodal organs with or without associated lymph nodeinvolvement (“Lymphoid neoplasms,” In American Joint Committee onCancer: AJCC Cancer Staging Manual 6th ed. New York, N.Y.: Springer,2002, pp. 393-406). Rituximab has been shown effective in treatingindolent and follicular lymphomas (Boye et al., Annals of Oncol.14:520-535, 2003).

d. Cervical Cancer

The cervix is the neck of the uterus that opens into the vagina.Cervical cancer, also called cervical carcinoma, develops from abnormalcells on the surface of the cervix. Cervical cancer is one of the mostcommon cancers affecting women. Cervical cancer is usually preceded bydysplasia, precancerous changes in the cells on the surface of thecervix. These abnormal cells can progress to invasive cancer. Once thecancer appears it can progress through four stages. The stages aredefined by the extent of spread of the cancer. The more the cancer hasspread, the more extensive the treatment is likely to be. There are 2main types of cervical cancer: (1) Squamous type (epidermoid cancer):This is the most common type, accounting for about 80% to 85% ofcervical cancers. This cancer may be caused by sexually transmitteddiseases. One such sexual disease is the human papillomavirus, whichcauses venereal warts. The cancerous tumor grows on and into the cervix.This cancer generally starts on the surface of the cervix and may bediagnosed at an early stage by a Pap smear. (2) Adenocarcinoma: Thistype of cervical cancer develops from the tissue in the cervical glandsin the canal of the cervix. Early cervical cancer usually causes nosymptoms. The cancer is usually detected by a Pap smear and pelvic exam.Later stages of cervical cancer cause abnormal vaginal bleeding or abloodstained discharge at unexpected times, such as between menstrualperiods, after intercourse, or after menopause. Abnormal vaginaldischarge may be cloudy or bloody or may contain mucus with a bad odor.Advanced stages of the cancer may cause pain (University of MichiganHealth System Internet website). The effects of an agent (e.g., ananti-zB7H6 antibody-drug conjugate) on tumor response can be evaluatedin a human tumor xenograft model similar to that described in Downs etal., Gynecol. Oncol. 98:203-10, 2005; and Li et al., Int. J. Gynecol.Cancer 15:301-7, 2005.

e. Head and Neck Tumors

Most cancers of the head and neck are of a type called carcinoma (inparticular squamous cell carcinoma). Carcinomas of the head and neckstart in the cells that form the lining of the mouth, nose, throat orear, or the surface layer covering the tongue. However, cancers of thehead and neck can develop from other types of cells. Lymphoma developsfrom the cells of the lymphatic system. Sarcoma develops from thesupportive cells which make up muscles, cartilage or blood vessels.Melanoma starts from cells called melanocytes, which give colour to theeyes and skin. The symptoms of a head and neck cancer will depend onwhere it is—for example, cancer of the tongue may cause some slurring ofspeech. The most common symptoms are an ulcer or sore area in the heador neck that does not heal within a few weeks; difficulty in swallowing,or pain when chewing or swallowing; trouble with breathing or speaking,such as persistent noisy breathing, slurred speech or a hoarse voice; anumb feeling in the mouth; a persistent blocked nose, or nose bleeds;persistent earache, ringing in the ear, or difficulty in hearing; aswelling or lump in the mouth or neck; pain in the face or upper jaw; inpeople who smoke or chew tobacco, pre-cancerous changes can occur in thelining of the mouth, or on the tongue. These can appear as persistentwhite patches (leukoplakia) or red patches (erythroplakia). They areusually painless but can sometimes be sore and may bleed (CancerbacupInternet website). The effects of an agent (e.g., an anti-zB7H6antibody-drug conjugate) on tumor response can be evaluated in a humanhead and neck tumor xenograft model similar to that described inKuriakose et al., Head Neck 22:57-63, 2000; Cao et al., Clin. CancerRes. 5:1925-34, 1999; Braakhuis et al., Cancer Res. 51:211-4, 1991; andBaker, Laryngoscope 95:43-56, 1985.

f. Brain Cancer

Tumors that begin in brain tissue are known as primary tumors of thebrain. Primary brain tumors are named according to the type of cells orthe part of the brain in which they begin. The most common primary braintumors are gliomas. They begin in glial cells. There are many types ofgliomas. (1) Astrocytoma—The tumor arises from star-shaped glial cellscalled astrocytes. In adults, astrocytomas most often arise in thecerebrum. In children, they occur in the brain stem, the cerebrum, andthe cerebellum. A grade III astrocytoma is sometimes called ananaplastic astrocytoma. A grade IV astrocytoma is usually called aglioblastoma multiforme. (2) Brain stem glioma—The tumor occurs in thelowest part of the brain. Brain stem gliomas most often are diagnosed inyoung children and middle-aged adults. (3) Ependymoma—The tumor arisesfrom cells that line the ventricles or the central canal of the spinalcord. They are most commonly found in children and young adults. (4)Oligodendroglioma—This rare tumor arises from cells that make the fattysubstance that covers and protects nerves. These tumors usually occur inthe cerebrum. They grow slowly and usually do not spread intosurrounding brain tissue. They are most common in middle-aged adults.The symptoms of brain tumors depend on tumor size, type, and location.Symptoms may be caused when a tumor presses on a nerve or damages acertain area of the brain. They also may be caused when the brain swellsor fluid builds up within the skull. These are the most common symptomsof brain tumors: Headaches (usually worse in the morning); Nausea orvomiting; Changes in speech, vision, or hearing; Problems balancing orwalking; Changes in mood, personality, or ability to concentrate;Problems with memory; Muscle jerking or twitching (seizures orconvulsions); and Numbness or tingling in the arms or legs (NationalCancer Institute's Internet website). The effects of an agent (e.g., ananti-zB7H6 antibody-drug conjugate) on tumor response can be evaluatedin a human glioma xenograft model similar to that described in Bello etal., Clin. Cancer Res. 8:3539-48, 2002.

g. Thyroid Cancer

Papillary and follicular thyroid cancers account for 80 to 90 percent ofall thyroid cancers. Both types begin in the follicular cells of thethyroid. Most papillary and follicular thyroid cancers tend to growslowly. If they are detected early, most can be treated successfully.Medullary thyroid cancer accounts for 5 to 10 percent of thyroid cancercases. It arises in C cells, not follicular cells. Medullary thyroidcancer is easier to control if it is found and treated before it spreadsto other parts of the body. Anaplastic thyroid cancer is the leastcommon type of thyroid cancer (only 1 to 2 percent of cases). It arisesin the follicular cells. The cancer cells are highly abnormal anddifficult to recognize. This type of cancer is usually very hard tocontrol because the cancer cells tend to grow and spread very quickly.Early thyroid cancer often does not cause symptoms. But as the cancergrows, symptoms may include: A lump, or nodule, in the front of the necknear the Adam's apple; Hoarseness or difficulty speaking in a normalvoice; Swollen lymph nodes, especially in the neck; Difficultyswallowing or breathing; or Pain in the throat or neck (National CancerInstitute's Internet website). The effects of an agent (e.g., ananti-zB7H6 antibody-drug conjugate) on tumor response can be evaluatedin a human tumor xenograft model similar to that described in Quidvilleet al., Endocrinology 145:2561-71, 2004.

h. Liver Cancer

There are two different types of primary liver cancer. The most commonkind is called hepatoma or hepatocellular carcinoma (HCC), and arisesfrom the main cells of the liver (the hepatocytes). This type is usuallyconfined to the liver, although occasionally it spreads to other organs.It occurs mostly in people with a liver disease called cirrhosis. Thereis also a rarer sub-type of hepatoma called Fibrolamellar hepatoma,which may occur in younger people and is not related to previous liverdisease. The other type of primary liver cancer is calledcholangiocarcinoma or bile duct cancer, because it starts in the cellslining the bile ducts. Most people who develop hepatoma usually alsohave a condition called cirrhosis of the liver. This is a fine scarringthroughout the liver which is due to a variety of causes includinginfection and heavy alcohol drinking over a long period of time.However, only a small proportion of people who have cirrhosis of theliver develop primary liver cancer. Infection with either the hepatitisB or hepatitis C virus can lead to liver cancer, and can also be thecause of cirrhosis, which increases the risk of developing hepatoma.People who have a rare condition called haemochromatosis, which causesexcess deposits of iron in the body, have a higher chance of developinghepatoma. A zB7H6-related agent of the present invention (e.g., asoluble zB7H6 polypeptide or an anti-zB7H6 antibody-drug conjugate) maybe used to treat, prevent, inhibit the progression of, delay the onsetof, and/or reduce the severity or inhibit at least one of the conditionsor symptoms associated with hepatocellular carcinoma. The hepatocellularcarcinoma may or may not be associated with a hepatitis (e.g., hepatitisA, hepatitis B, hepatitis C and hepatitis D) infection. The effects ofan agent (e.g., an anti-zB7H6 antibody-drug conjugate) on tumor responsecan be evaluated in a human tumor xenograft model similar to thatdescribed in Zhou et al., Clin. Cancer Res. 9:6030-7, 2003; and Huynh etal., J. Cell Mol. Med. 2008 (E-Published as a “Postprint,”10.1111/j.1582-4934.2008.00364.x, 2008, at Blackwell Synergy website).

i. Lung Cancer

The effects of an agent (e.g., an anti-zB7H6 antibody-drug conjugate) ontumor response can be evaluated in a human small/non-small cell lungcarcinoma xenograft model. Briefly, human tumors are grafted intoimmunodeficient mice and these mice are treated with an agent, such asan anti-zB7H6 antibody-drug conjugate, alone or in combination withother agents. Efficacy of the treatment can be demonstrated byevaluating tumor growth (Nemati et al., Clin Cancer Res. 6:2075-86,2000; and Hu et al., Clin. Cancer Res. 10:7662-70, 2004).

2. Endpoints and Anti-Tumor Activity for Solid Tumors

While each protocol may define tumor response assessments differently,exemplary guidelines can be found in Clinical Research Associates Manual(Southwest Oncology Group, CRAB, Seattle, Wash., Oct. 6, 1998, updatedAugust 1999) (“CRA Manual”). According to the CRA Manual (see chapter 7,“Response Accessment”), tumor response means a reduction or eliminationof all measurable lesions or metastases. Disease is generally consideredmeasurable if it comprises bi-dimensionally measurable lesions withclearly defined margins by medical photograph or X-ray, computerizedaxial tomography (CT), magnetic resonance imaging (MRI), or palpation.Evaluable disease means the disease comprises uni-dimensionallymeasurable lesions, masses with margins not clearly defined, lesion withboth diameters less than 0.5 cm, lesions on scan with either diametersmaller than the distance between cuts, palpable lesions with diameterless than 2 cm, or bone disease. Non-evaluable disease includes pleuraleffusions, ascites, and disease documented by indirect evidence.Previously radiated lesions that have not progressed are also generallyconsidered non-evaluable.

The criteria for objective status are required for protocols to assesssolid tumor response. Representative criteria include the following: (1)Complete Response (CR), defined as complete disappearance of allmeasurable and evaluable disease; no new lesions; no disease relatedsymptoms; no evidence of non-evaluable disease; (2) Partial Response(PR) defined as greater than or equal to 50% decrease from baseline inthe sum of products of perpendicular diameters of all measurablelesions; no progression of evaluable disease; no new lesions; applies topatients with at least one measurable lesion; (3) Progression, definedas 50% or an increase of 10 cm² in the sum of products of measurablelesions over the smallest sum observed using same techniques asbaseline, or clear worsening of any evaluable disease, or reappearanceof any lesion which had disappeared, or appearance of any new lesion, orfailure to return for evaluation due to death or deteriorating condition(unless unrelated to this cancer); (4) Stable or No Response, defined asnot qualifying for CR, PR, or Progression. (See Clinical ResearchAssociates Manual, supra.)

Additional endpoints that are accepted within the oncology art includeoverall survival (OS), disease-free survival (DFS), objective responserate (ORR), time to progression (TTP), and progression-free survival(PFS) (see Guidance for Industry: Clinical Trial Endpoints for theApproval of Cancer Drugs and Biologics, April 2005, Center for DrugEvaluation and Research, FDA, Rockville, Md.)

3. Combination Cancer Therapy

As previously discussed, in certain embodiments, a zB7H6 polypeptide,antibody, or other zB7H6-related agent is used in combination with asecond agent for treatment of a disease or disorder. When used fortreating cancer, a zB7H6 polypeptide, antibody, or other agent of thepresent invention, including, for example, an anti-zB7H6 antibody-drugconjugate, may be used in combination with conventional cancer therapiessuch as, e.g., surgery, radiotherapy, chemotherapy, or combinationsthereof. In certain aspects, other therapeutic agents useful forcombination cancer therapy with a zB7H6-related agent in accordance withthe present invention include anti-angiogenic agents. In some otheraspects, other therapeutic agents useful for combination therapy includean antagonist of certain factors that are involved in tumor growth suchas, for example, EGFR, ErbB2 (Her2), ErbB3, ErbB4, or TNF. In someaspects, an agent in accordance with the present invention isco-administered with a cytokine (e.g., a cytokine that stimulates animmune response against a tumor). Exemplary combination therapiesparticularly amenable for treatment of cancer are described in furtherdetail below.

a. Antibodies Targeting Tumor-Associated Antigens

As previously noted, antibody therapy has been particularly successfulin cancer treatment because certain tumors either display uniqueantigens, lineage-specific antigens, or antigens present in excessamounts relative to normal cells. One of the mechanisms associated withthe anti-tumor activity of monoclonal antibody therapy is antibodydependent cellular cytotoxicity (ADCC). In ADCC, monoclonal antibodiesbind to a target cell (e.g., cancer cell) and specific effector cellsexpressing receptors for the monoclonal antibody (e.g., NK cells,monocytes, granulocytes) bind the monoclonal antibody/target cellcomplex resulting in target cell death. Accordingly, in certainvariations of the present invention, a zB7H6 polypeptide, antibody, orother zB7H6-related agent having efficacy against a cancer isco-administered with a monoclonal antibody against a tumor-associatedantigen. In those variations where an anti-zB7H6 antibody is utilized,either to induce anti-tumor activity via ADCC or CDC or, alternatively,in the context of an anti-zB7H6 antibody-drug conjugate, the monoclonalantibody used in the combination will be an antibody to a secondtumor-specific or tumor-associated antigen. The dose and schedule of theMAbs is based on pharmacokinetic and toxicokinetic properties ascribedto the specific antibody co-administered, and should optimize theseeffects, while minimizing any toxicity that may be associated withadministration of a zB7H6 polypeptide, antibody, or other zB7H6-relatedagent.

Combination therapy with a zB7H6-related agent as described herein and amonoclonal antibody against a tumor-associated antigen may be indicatedwhen a first line treatment has failed and may be considered as a secondline treatment. The present invention also provides using thecombination as a first line treatment in patient populations that arenewly diagnosed and have not been previously treated with anticanceragents (“de novo patients”) and patients that have not previouslyreceived any monoclonal antibody therapy (“naïve patients”).

A zB7H6-related agent as described herein is also useful in combinationtherapy with monoclonal antibodies against tumor-associated antigens inthe absence of any direct antibody-mediated ADCC or CDC of tumor cells.For example, antibodies that block an inhibitory signal in the immunesystem can lead to augmented immune responses. Examples include (1)antibodies against molecules of the B7R family that have inhibitoryfunction such as, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4),programmed death-1 (PD-1), B and T lymphocyte attenuator (BTLA); (2)antibodies against inhibitory cytokines like IL-10, TGFβ; and (3)antibodies that deplete or inhibit functions of suppressive cells likeanti-CD25 or CTLA-4. For example, anti-CTLA4 MAbs in both mice andhumans are thought to either suppress function of immune-suppressiveregulatory T cells (Tregs) or inhibit the inhibitory signal transmittedthrough binding of CTLA-4 on T cells to B7-1 or B7-2 molecules on APCsor tumor cells.

Table 6 is a non-exclusive list of monoclonal antibodies approved orbeing tested for which combination therapy in accordance with thepresent invention is possible.

TABLE 6 Monoclonal Antibody Therapies for Use in Combination with PDGFRβand/or VEGF-A Antagonists Clinical Target Drug Name Indication CompanyTRAIL-R1 HGS-ETR1 Cancers HGS TRAIL-R2 HGS-ETR2 solid tumors HGS CD40SGN40 MM Seattle Genetics HER2 Herceptin Breast cancer Genentech EGF-RABX-EGF CRC, NSCLC, Abgenix RCC EGF-R EMD72000 solid tumors Merck EGF-RMDX-214 EGF-R-positive Medarex tumors EGF-R Erbitux CRC Imclone α5β3integrin Vitaxin psoriasis, AME/Lilly prostate cancer CD152 CTLA-4Cancers Medarex CD49e Integrin α5 Cancers Protein Design Labs MUC18(TIM-like) ABX-MA1 Melanoma TAG-72 Mucin Anatumomab Cancers CD3Ecromeximab Melanoma Kyowa Hakko CD64 (Fc GR1) AntiCD64 Cancers MedarexCEA CEA-Cide Cancers Immunomedics EpCAM Panorex colorectal Centocorcancer Lewis-Y-Ag SGN15 Cancers Seattle Genetics

b. Tyrosine Kinase Inhibitors

In some embodiments, a zB7H6 polypeptide, antibody, or otherzB7H6-related agent as described herein is used in combination with atyrosine kinase inhibitor. Tyrosine kinases are enzymes that catalyzethe transfer of the γ phosphate group from the adenosine triphosphate totarget proteins. Tyrosine kinases can be classified as receptor andnonreceptor protein tyrosine kinases. They play an essential role indiverse normal cellular processes, including activation through growthreceptors and affect proliferation, survival and growth of various celltypes. Additionally, they are thought to promote tumor cellproliferation, induce anti-apoptotic effects and promote angiogenesisand metastasis. In addition to activation through growth factors,protein kinase activation through somatic mutation is a common mechanismof tumorigenesis. Some of the mutations identified are in B-Raf kinase,FLt3 kinase, BCR-ABL kinase, c-KIT kinase, epidermal growth factor(EGFR) and PDGFR pathways. The Her2, VEGFR and c-Met are othersignificant receptor tyrosine kinase (RTK) pathways implicated in cancerprogression and tumorigenesis. Because a large number of cellularprocesses are initiated by tyrosine kinases, they have been identifiedas key targets for inhibitors.

Tyrosine kinase inhibitors (TKIs) are small molecules that act insidethe cell, competing with adenosine triphosphate (ATP) for binding to thecatalytic tyrosine kinase domain of both receptor and non-receptortyrosine kinases. This competitive binding blocks initiation ofdownstream signaling leading to effector functions associated with thesesignaling events like growth, survival, and angiogenesis. Using astructure and computational approach, a number of compounds fromnumerous medicial chemistry combinatorial libraries was identified thatinhibit tyrosine kinases.

Most TKIs are thought to inhibit growth of tumors through directinhibition of the tumor cell or through inhibition of angiogenesis.Moreover, certain TKIs affect signaling through the VEGF familyreceptors, including sorafenib and sunitinib. In some cases TKIs havebeen shown to activate functions of dendritic cells and other innateimmune cells, like NK cells. This has been recently reported in animalmodels for imatinib. Imatinib is a TKI that has shown to enhance killeractivity by dendritic cells and NK cells (for review, see Smyth et al.,NEJM 354:2282, 2006).

BAY 43-9006 (sorafenib, Nexavar®) and SU11248 (sunitinib, Sutent®) aretwo such TKIs that have been recently approved for use in metastaticrenal cell carcinoma (RCC). A number of other TKIs are in late and earlystage development for treatment of various types of cancer. Other TKIsinclude, but are not limited to: Imatinib mesylate (Gleevec®, Novartis);Gefitinib (Iressa®, AstraZeneca); Erlotinib hydrochloride (Tarceva®,Genentech); Vandetanib (Zactima®, AstraZeneca), Tipifarnib (Zarnestra®,Janssen-Cilag); Dasatinib (Sprycel®, Bristol Myers Squibb); Lonafarnib(Sarasar®, Schering Plough); Vatalanib succinate (Novartis, ScheringAG); Lapatinib (Tykerb®, GlaxoSmithKline); Nilotinib (Novartis);Lestaurtinib (Cephalon); Pazopanib hydrochloride (GlaxoSmithKline);Axitinib (Pfizer); Canertinib dihydrochloride (Pfizer); Pelitinib(National Cancer Institute, Wyeth); Tandutinib (Millennium); Bosutinib(Wyeth); Semaxanib (Sugen, Taiho); AZD-2171 (AstraZeneca); VX-680(Merck, Vertex); EXEL-0999 (Exelixis); ARRY-142886 (Array BioPharma,AstraZeneca); PD-0325901 (Pfizer); AMG-706 (Amgen); BIBF-1120(Boehringer Ingelheim); SU-6668 (Taiho); CP-547632 (OSI); (AEE-788(Novartis); BMS-582664 (Bristol-Myers Squibb); JNK-401 (Celgene); R-788(Rigel); AZD-1152 HQPA (AstraZeneca); NM-3 (Genzyme Oncology); CP-868596(Pfizer); BMS-599626 (Bristol-Myers Squibb); PTC-299 (PTC Therapeutics);ABT-869 (Abbott); EXEL-2880 (Exelixis); AG-024322 (Pfizer); XL-820(Exelixis); OSI-930 (OSI); XL-184 (Exelixis); KRN-951 (Kirin Brewery);CP-724714 (OSI); E-7080 (Eisai); HKI-272 (Wyeth); CHIR-258 (Chiron);ZK-304709 (Schering AG); EXEL-7647 (Exelixis); BAY-57-9352 (Bayer);BIBW-2992 (Boehringer Ingelheim); AV-412 (AVEO); YN-968D1 (AdvenchenLaboratories); Midostaurin (Novartis); Perifosine (AEterna Zentaris,Keryx, National Cancer Institute); AG-024322 (Pfizer); AZD-1152(AstraZeneca); ON-01910Na (Onconova); and AZD-0530 (AstraZeneca).

c. Chemotherapy Combinations

In certain embodiments, a zB7H6 polypeptide, antibody, or otherzB7H6-related agent is administered in combination with one or morechemotherapeutic agents. Chemotherapeutic agents have different modes ofactions, for example, by influencing either DNA or RNA and interferingwith cell cycle replication. Examples of chemotherapeutic agents thatact at the DNA level or on the RNA level are anti-metabolites (such asAzathioprine, Cytarabine, Fludarabine phosphate, Fludarabine,Gemcitabine, cytarabine, Cladribine, capecitabine 6-mercaptopurine,6-thioguanine, methotrexate, 5-fluoroouracil and hyroxyurea; alkylatingagents (such as Melphalan, Busulfan, Cis-platin, Carboplatin,Cyclophosphamide, Ifosphamide, Dacarabazine, Procarbazine, Chlorambucil,Thiotepa, Lomustine, Temozolamide); anti-mitotic agents (such asVinorelbine, Vincristine, Vinblastine, Docetaxel, Paclitaxel);topoisomerase inhibitors (such as Doxorubincin, Amsacrine, Irinotecan,Daunorubicin, Epirubicin, Mitomycin, Mitoxantrone, Idarubicin,Teniposide, Etoposide, Topotecan); antibiotics (such as actinomycin andbleomycin); asparaginase; anthracyclines or taxanes.

d. Radiotherapy Combinations

In some variations, a zB7H6 polypeptide, antibody, or otherzB7H6-related agent is administered in combination with radiotherapy.Certain tumors can be treated with radiation or radiopharmaceuticals.Radiation therapy is generally used to treat unresectable or inoperabletumors and/or tumor metastases. Radiotherapy is typically delivered inthree ways. External beam irradiation is administered at distance fromthe body and includes gamma rays (⁶⁰Co) and X-rays. Brachytherapy usessources, for example ⁶⁰Co, ¹³⁷Cs, ¹⁹²Ir, or ¹²⁵I, with or in contactwith a target tissue.

e. Hormonal Agent Combinations

In some embodiments, a zB7H6 polypeptide, antibody, or otherzB7H6-related agent is administered in combination with a hormone oranti-hormone. Certain cancers are associated with hormonal dependencyand include, for example, ovarian cancer, breast cancer, and prostatecancer. Hormonal-dependent cancer treatment may comprise use ofanti-androgen or anti-estrogen compounds. Hormones and anti-hormonesused in cancer therapy include Estramustine phosphate, Polyestradiolphosphate, Estradiol, Anastrozole, Exemestane, Letrozole, Tamoxifen,Megestrol acetate, Medroxyprogesterone acetate, Octreotide, Cyproteroneacetate, Bicaltumide, Flutamide, Tritorelin, Leuprorelin, Buserelin andGoserelin.

VII. Methods of Screening

In another aspect, the present invention provides methods of screeningfor an agonsist or antagonstist of the interaction of zB7H6 with NKp30.Generally, such methods of screening for an antagonist include thefollowing steps: (a) contacting an agent with a zB7H6 polypeptide in thepresence of an NKp30 polypeptide; (b) detecting a measure of theinteraction of the zB7H6 polypeptide with the NKp30 polypeptide; and (c)determining whether the level of the zB7H6/NKp30 interaction measured instep (b) is significantly less relative to the level of interactionmeasured for control zB7H6 and NKp30 polypeptides in the absence of theagent, such that if the level of zB7H6/NKp30 interaction is less, thenthe agent is identified as an antagonist of the interaction of zB7H6with NKp30.

Methods of screening for an agonist generally include the followingsteps:

(a) contacting an agent with a zB7H6 polypeptide in the presence of anNKp30 polypeptide; (b) detecting a measure of the interaction of thezB7H6 polypeptide with the NKp30 polypeptide; and (c) determiningwhether the level of the zB7H6/NKp30 interaction measured in step (b) issignificantly greater relative to the level of interaction measured forcontrol zB7H6 and NKp30 polypeptides in the absence of the agent, suchthat if the level of zB7H6/NKp30 interaction is greater, then the agentis identified as an agonist of the interaction of zB7H6 with NKp30.

A measure of zB7H6 interaction with NKp30 can include, for example,detection of zB7H6 binding to NKp30 as well as the ability of the zB7H6polypeptide to trigger NKp30-mediated cellular activity (e.g., cytolyticactivity), or of the ability of the NKp30 polypeptide to triggerzB7H6-mediated cellular activity. For identifying agonists of thezB7H6/NKp30 interaction, particularly where the measure of theinteraction is a level of NKp30- or zB7H6-mediated cellular activity,the methods can further include an additional control to determinewhether the agent is capable of inducing the cellular activity in theabsence of the zB7H6 polypeptide or NKp30 polypeptide, such that if theagent is capable of inducing the cellular activity in the absence of thezB7H6 polypeptide or NKp30 polypeptide, then the agent is not an agonistof the interaction of zB7H6 with NKp30.

zB7H6 polypeptides for use in the screening methods will generallycomprise a zB7H6 extracellular domain, or a functional variant orfragment thereof. Accordingly, a zB7H6 polypeptide for use in screeningwill include a polypeptide region selected from the following:

-   -   (i) the extracellular domain of the zB7H6 polypeptide of SEQ ID        NO:2 (i.e., residues 25-266 of SEQ ID NO:2);    -   (ii) a functional variant of the zB7H6 extracellular domain of        (i), the variant having at least 80% identity with residues        25-266 of SEQ ID NO:2; and    -   (iii) a functional fragment of the zB7H6 extracellular domain        of (i) or of the domain variant of (ii).

In typical variations, the zB7H6 polypeptide includes the extracellulardomain of the zB7H6 SEQ ID NO:2 (i.e., residues 25-266 of SEQ ID NO:2),or a functional variant having at least 90% or 95% sequence identitywith residues 25-266 of SEQ ID NO:2. The zB7H6 polypeptide can be asoluble zB7H6 receptor as disclosed herein. In alternative variations,the zB7H6 polypeptide is a membrane-bound form of zB7H6 expressed on acell (e.g., a zB7H6 polypeptide having a GPI linkage, or a zB7H6polypeptide having a functional transmembrane domain, such as the zB7H6polypeptide of SEQ ID NO:2, expressed on a recombinant cell).

Similarly, the NKp30 polypeptide for use in the screening method willinclude the extracellular domain of NKp30, or a functional variant orfragment thereof. Typically, the NKp30 polypeptide is a human NKp30polypeptide or a polypeptide derived from human NKp30. The NKp30polypeptide can be a soluble NKp30 receptor or a membrane-bound form ofNKp30. In certain variations, the NKp30 is a full-length NKp30 protein(e.g., full-length human NKp30) expressed on cells; such embodiments areparticularly amenable to, inter alia, use of NKp30-mediated cytolyticactivity as a functional read-out to detect the interaction of zB7H6with NKp30.

In certain variations utilizing zB7H6 or NKp30 polypeptides expressed onrecombinant cells, a cDNA or gene encoding the zB7H6 or NKp30 receptoris combined with other genetic elements required for its expression(e.g., a transcription promoter), and the resulting expression vector isinserted into a host cell. Cells that express the DNA and producefunctional receptor are selected and used within a variety of screeningsystems. Each component of the monomeric, homodimeric, heterodimeric andmultimeric receptor complex can be expressed in the same cell. Moreover,the components of the monomeric, homodimeric, heterodimeric andmultimeric receptor complex can also be fused to a transmembrane domainor other membrane fusion moiety to allow complex assembly and screeningof transfectants. In some embodiments, each of the zB7H6 polypeptide andNKp30 polypeptide are expressed in separate host cells. Alternatively,only one of the zB7H6 and NKp30 polypeptides is expressed in a cell.

In an animal model system, the cell can be contacted with the candidateagent by administering the candidate agent to the animal. The candidateagent can be administered orally, intravenously, by infusion orinjection, or the like.

Agents for use in screening can include any agent with a potential tostructurally interact with biomolecules, particularly proteins, throughnon-covalent interactions, such as, for example, through hydrogen bonds,ionic bonds, van der Waals attractions, or hydrophobic interactions.Accordingly, many types of agents can be screened by the presentmethods. Suitable candidate agents include, for example, smallmolecules, nucleic acids, peptides, peptidomimetics, syntheticcompounds, and/or natural compounds.

Agents for screening can include random and/or semi-random libraries ofpeptides and/or nucleic acids. In variations comprising expression ofrecombinant zB7H6 or NKp30 in host cells, a nucleic acid agent can bescreened by contacting the cell of the expression system with thenucleic acid. In a specific example, a genomic or cDNA library can beintroduced into and expressed in a population of recombinant cellsexpressing zB7H6 or NKp30 to identify a genetic agent that reduces orenhances the interaction of zB7H6 with NKp30.

In other embodiments, an agent to be screened is a peptidomimetic. Theterm “peptidomimetic” refers to a synthetic chemical compound that hassubstantially the same structural and functional characteristics as aprotein, polypeptide, or peptide. Peptide analogs are commonly used inthe pharmaceutical industry as non-peptide drugs with propertiesanalogous to those of the template peptide. These types of non-peptidecompounds are termed “peptide mimetics” or “peptidomimetics” (see, e.g.,Fauchere, J. Adv. Drug Res. 15:29, 1986; Veber and Freidinger TINS p.392, 1985; and Evans et al., J. Med. Chem. 30:1229, 1987). Peptidemimetics that are structurally similar to therapeutically usefulpeptides may be used to produce an equivalent or enhanced therapeutic orprophylactic effect. Generally, peptidomimetics are structurally similarto a paradigm polypeptide (e.g., a polypeptide that has a desiredbiological or pharmacological activity), but have one or more peptidelinkages optionally replaced by a linkage selected from the groupconsisting of, e.g., —CH2NH—, —CH2S—, —CH2—CH2—, —CH.═CH—(cis andtrans), —COCH2—, —CH(OH)CH2—, and —CH2SO—. The mimetic can be eitherentirely composed of synthetic, non-natural analogues of amino acids,or, is a chimeric molecule of partly natural peptide amino acids andpartly non-natural analogs of amino acids. The mimetic can alsoincorporate natural amino acid conservative substitutions as long assuch substitutions also do not substantially alter the mimetic'sstructure and/or activity.

Agents for screening can also be from libraries of synthetic and/ornatural compounds. One example is a library of FDA-approved compoundsthat can be used by humans. In addition, synthetic compound librariesare commercially available from a number of companies includingMaybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton,N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (NewMilford, Conn.), and a rare chemical library is available from Aldrich(Milwaukee, Wis.).

Combinatorial libraries are available and/or can be prepared.Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant, and animal extracts are also available, for example, fromPan Laboratories (Bothell, Wash.) or MycoSearch (N.C.), or can beprepared. Compounds isolated from natural sources, such as animals,bacteria, fungi, plant sources, including leaves and bark, and marinesamples also can be screened as candidate agents.

Other suitable agents include antisense molecules, ribozymes, andantibodies (including single chain antibodies and Fv fragments). Forexample, an antisense molecule that binds to a translational ortranscriptional start site, or a splice junction, can be a candidateagent. Additionally, natural and synthetically-produced libraries andcompounds are readily modified through conventional chemical, physical,and biochemical means.

Screening of such libraries, including combinatorially generatedlibraries (e.g., peptide libraries) can be performed in a rapid andefficient way to screen a large number of related and/or unrelatedcompounds. Combinatorial approaches also lend themselves to rapidevolution of potential therapeutic agents by the creation of second,third and fourth generation compounds modeled on active, but otherwiseundesirable compounds.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175; Furka, Int. J. Pept. Prot. Res. 37:487-93,1991; Houghton et al., Nature 354:84-88, 1991). Other chemistries forgenerating chemical diversity libraries also can be used. Suchchemistries include, but are not limited to: peptoids (see, e.g., PCTPublication No. WO 91/19735), encoded peptides (see, e.g., PCTPublication WO 93/20242), random bio-oligomers (see, e.g., PCTPublication No. WO 92/00091), benzodiazepines (see, e.g., U.S. Pat. No.5,288,514; Baum, C&EN, Jan. 18, 1993, p. 33), diversomers such ashydantoins, benzodiazepines and dipeptides (see, e.g., Hobbs et al.,Proc. Nat. Acad. Sci. USA 90:6909-13, 1993), vinylogous polypeptides(see, e.g., Hagihara et al., J. Amer. Chem. Soc. 114:6568, 1992),nonpeptidal peptidomimetics with glucose scaffolding (see, e.g.,Hirschmann et al., J. Amer. Chem. Soc. 114:9217-18, 1992), analogousorganic syntheses of small compound libraries (see, e.g., Chen et al.,J. Amer. Chem. Soc. 116:2661, 1994), oligocarbamates (see, e.g., Cho etal., Science 261:1303, 1993), peptidyl phosphonates (see, e.g., Campbellet al., J. Org. Chem. 59:658, 1994), nucleic acid libraries (see, e.g.,Ausubel et al., supra; Sambrook, supra), peptide nucleic acid libraries(see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g.,Vaughn et al., Nature Biotechnology, 14:309-14, 1996 andPCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al.,Science 274:1520-22, 1996; U.S. Pat. No. 5,593,853), small organicmolecule libraries, such as isoprenoids (see, e.g., U.S. Pat. No.5,569,588), thiazolidinones and metathiazanones (see, e.g., U.S. Pat.No. 5,549,974), pyrrolidines (see, e.g., U.S. Pat. Nos. 5,525,735 and5,519,134), morpholino compounds (see, e.g., U.S. Pat. No. 5,506,337),or the like.

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy.; Symphony, Rainin, Woburn, Mass.; 433A Applied Biosystems, FosterCity, Calif.; 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J.; Tripos, Inc., St. Louis, Mo.; 3DPharmaceuticals, Exton, Pa.; Martek Biosciences, Columbia, Md.; etc.).

Agents that are initially identified by any of the foregoing screeningmethods can be further tested to validate the apparent activity. Forexample, subsequent validation can be performed with suitable animalmodels or ex vivo human cells. For in vivo validation using an animalmodel system, the basic format of such methods can involve administeringan agent identified during an initial screen to an animal that serves asa model for an NK cell-associated disease or disorder and thendetermining if NK cell activity is modulated, or if other clinicalsymptoms of the disease or disorder are ameliorated. The animal modelsutilized in validation studies generally are mammals of any kind.Specific examples of suitable animals include, but are not limited to,primates, mice, and rats.

The invention is further illustrated by the following non-limitingexamples.

Example 1 Inhibition of NK-92 Cytolytic Activity Against K562 Targetswith Soluble NKp30/VASP

A cytolytic assay was performed with NK-92 cells as effectors againstK562 targets.

NK-92 cells were washed 1× with HBSSF (Hank's buffered saline (Ca, MgFree)+5% FBS) and resuspended in HBSSF at 1.35×10⁶/ml (to achieve a 27:1ratio). 150 μl of washed cells were plated in the top row of a U-bottom96-well plate and serially diluted (1:3) into HBSSF.

K562 target cells were washed 1× with HBSSF and labeled at 1×10⁶cells/ml in 10 μM calcein AM Molecular probes #C1430 (2.5 μl/ml of 4 mMstock in DMSO, 4 mM=4 mg/ml) for 60 minutes at 37° C. Labeled cells werewashed 2× in HBSSF and 1×10⁶ cells were resuspended in 20 ml HBSSF (5000cells/100 μl). 100 ul of suspended target cells were added to dilutedeffectors for a total volume of 200 ul. A soluble form of NKp30(NKp30/VASP A1683F) was also added to some sets of serially dilutedwells at a concentration of 2 μg/ml.

Effector and target cells were spun at 500 rpm for 2 min, incubated at37° C. for 3 hours, spun at 1500 rpm for 5 min, and then 100 μlsupernatant transferred to a new flat bottom 96-well. Flat bottom platescontaining transferred supernatants were read on a fluoremeter for 1second at an excitation wavelength of 485 nm and emission wavelength of535 nm.

As shown in FIG. 1, soluble NKp30/VASP A1683F inhibited the cytolyticactivity of NK-92 cells against K562 targets. (See FIG. 1A.) Other VASPcontrols had no effect, suggesting that the ability of NK-92 to lyseK562 targets was dependent on NKp30.

In a separate cytolytic assay experiment, soluble NKp30/VASP was addedto wells containing NK-92 effectors and K562 targets (effector:targetratio of 9:1) at different concentrations (0.25, 0.5, 1.0, 2.0, 4.0,8.0, and 16.0 μg/ml). The results of this experiment demonstrated thatsoluble NKp30 inhibits lysis by NK-92 cells in a dose dependent manner.(See FIG. 1B.) These results suggested the presence of a ligand forNKp30 on K562 cells and encouraged further investigation.

Example 2 Soluble NKp30 Specifically Binds K562 Cells

K562 cells were probed by FACS with a soluble form of NKp30 (NKp30/mFc2(SEQ ID NO:8), containing the extracellular domain of NKp30 and a murineFc fragment). K562 cells were resuspended in PBS/2% FBS at aconcentration of 1.6×10⁶ cells/ml (160,000 cells/sample). 100 μl sampleswere aliquoted and 1 μl of whole human IgG (Jackson #009-000-003) addedto each. NKp30/mFc2 probe was added at a concentration of 2 μg/mltogether with 10 μg/ml Heparin and 100-fold mass excess of a VASPprotein (NKp30/VASP or a control VASP protein, human zB7R1/VASP (SEQ IDNO:12) or B7-DC/VASP (SEQ ID NO:13)). Cells were incubated for 1 hour onice and washed with 2 ml cold PBS. Washed cells were resuspended in 100μl of PBS/2% FBS with 1 μl PE anti-mIgG (Jackson 115-116-071) andincubated for 30 minutes on ice. Cells were then washed twice with 2 mlcold PBS, resuspended in 500 μl of PBS, and analyzed for PE staining ona FACSCalibur.

As shown in FIG. 2, NKp30/mFc2 bound to K562 cells (“No Comp.”). Thisbinding was competable with NKp30/VASP, but not competable with controlVASP proteins (“hzB7R1/Vasp” and “B7-DC/Vasp”), demonstrating that thebinding of NKp30/mFc2 to K562 cells was specific.

In a separate FACS experiment, K562 cells and BaF3 cells were probedwith NKp30/mFc2 conjugated to biotin (NKp30/mFc2-biotin, 4 μg/ml). Forthese studies, PE-conjugated streptavidin (BD Pharmingen 554061) wasused as the secondary reagent. The results of this experimentdemonstrated that NKp30/mFc2 bound to K562 cells, but not to BaF3 cells.(See FIG. 3.)

Example 3 Crosslinking of K562 Cells and Biotinylated NKp30/mFc2

In an effort to identify an NKp30 ligand on K562 cells, K562 cells werecross-linked with biotinylated NKp30/mFc2, followed byimmunoprecipitation and mass spectrometry.

Four samples, the sample of interest and three negative control samples,were analyzed. The sample of interest was K562 cells incubated withbiotinylated NKp30/mFc2. The three negative control samples were K562cells with no NKp30 and BaF3 cells with and without NKp30. 100×10⁶ cellswere washed once in PBS and resuspended in 2 ml binding buffer (RPMI, 3mg/ml BSA, 20 mM HEPES), in the presence or absence of 2 μg/mlNKp30/mFc2-biotin, and incubated for 2 hours on ice. Cells were washed(once in binding buffer, once in PBS), resuspended in 1 ml ofcrosslinking reagent (3 mm BS³ [Pierce 21580]), and incubated for 30minutes at room temperature. 7.5 μl of 2 M Tris (pH 7.4) was then addedfor a final Tris concentration 15 mM, and cells were incubated for 15minutes at room temperature. Cells were washed twice in PBS and thenlysed in 1 ml RIPA/1% TX-100/0.1% SDS for 5 minutes on ice (RIPA buffer:20 mM Tris pH 7.4, 150 mM NaCl, 2 mM EGTA, 1 mM NaV0₄, 1 mMβ-glycerophosphate, 1 tablet/25 mls Complete Mini Protease inhibitorcocktail tablet (Roche 10946900)). Lysate supernatants were incubatedwith 50 μl of streptavidin agarose (Pierce 20347), lysate supernatantsfor 2 hours at 4° C. with rocking. Streptavidin agarose was washed threetimes in PBS. Bound protein was eluted by resuspension of strepatividinagarose in 7.5 μl Nupage sample buffer (Invitrogen NP0007), 19.5 μl H₂0and 3 μl reducing agent (Invitrogen NP0004) followed by boiling for 10minutes. Samples were then split in half and each half run on one of twoidentical 4-12% NuPage gels (about 40×10⁶ cells/lane). UncrosslinkedNKp30/mFc2-biotin (100 ng, 33 ng, 11 ng, and 3.6 ng) was also run onthese gels as a control. One of the two gels was used for tandem massspectrometry analysis while the other was used for Western blotanalysis.

For Western blot analysis, proteins were transferred to a nitrocellulosemembrane (Invitrogen LC2000) in Western transfer buffer (0.025 MTris/0.186 M glycine/20% (v/v) methanol) at 600 mAmps constant currentfor 45 minutes. The nitrocellulose membrane was then blocked withblocking buffer Western A (0.097% Tris Base (w/w)/0.661% Tris HCl(w/w)/0.186% EDTA (w/w)/0.05% Igepal (v/w)/0.877% NaCl (w/w)/0.25%gelatin 1 (w/w)) for 1 hour at room temperature. Blocked membrane wasprobed with Streptavidin-HRP (1:8000, Pierce 21126) for 1 hour at roomtemperature and then washed three times with PBS. Washed membrane wasincubated in 10 ml ECL A+B buffer (Amersham RPN2209) for 1 min at roomtemperature, wrapped in Saran® wrap, and exposed to x-ray film. A 5second exposure gave the result shown in FIG. 4. As shown in FIG. 4,high molecular weight signal is only detected for K562 cells probed withNKp30/mFc2-biotin. Proteins corresponding to this high molecular weightband were excised from the corresponding NuPage gel for tandem massspectrometry analysis.

Example 4 Identification of zB7H6 by LC-MS/MS Proteomic Analysis ofNKp30 Interacting Proteins Introduction

K562 cells were incubated with biotinylated NKp30/mFc2 and anyinteractions were preserved by covalently binding the interaction with achemical crosslinker (see Example 3, supra). Differential massspectrometry analysis can identify unique proteins by using an automatedsearch algorithm to match tandem mass spectra with peptide sequences. Inthis analysis, the search algorithm X!Tandem was used to identifyproteins unique to the interaction of NKp30/mFc2 with K562 cells.

Materials and Methods

Four samples, the sample of interest and three negative control samples,were analyzed. The sample of interest was K562 cells incubated withbiotinylated NKp30/mFc2. The three negative control samples were K562cells with no NKp30 and BaF3 cells with and without NKp30. Each samplewas reacted with a chemical crosslinker to covalently link anyprotein-protein interactions and the biotinylated components wereseparated and collected by precipitating with streptavidin agarose.

The streptavidin purified fractions containing the biotinylatedcomponents were separated by SDS-PAGE electrophoresis. A Western blotwas prepared and probed with Strepavidin-HRP (see Example 3, supra). Thesecond gel was coomassie stained. FIGS. 5A and 5B show thecoomassie-stained gel and corresponding Western blot juxtaposed.

16 gel bands were excised. These bands corresponded to regions 11-14,21-24, 31-34, and 41-44 as delineated in FIG. 5A. The proteins in thesegel bands were reduced with TCEP (25 μl, 25 mM, 80° C., 15 min), theresulting free cysteines were capped with IAM (25 μl, 100 mM, 25° C., 2hr) and the sample was digested with trypsin (Promega V5111, lot18889904, 10 μl, 20 μg/mL, 37° C., 18 hr). The resulting peptides wereextracted from the gel pieces, dried down and reconstituted in 20 μL of0.1% FA. 5 μl of the resulting peptide mixture was separated on MagicC18AQ 3 μm, 200A resin packed into ˜10 cm of 50 um fused silica. Elutingpeptides were analyzed on an LTQ Ion Trap mass spectrometer. Theanalysis on the mass spectrometer consisted of a cycle of ten scans. Inthe first scan, a full MS scan from 400 to 2000 m/z was obtained.Subsequent scans analyzed the nine most intense ions by MS/MS. Dynamicexclusion prevented an analyzed ion from being targeted for MS/MSanalysis from 15 seconds to 30 seconds after its initial MS/MS analysis.

The raw data files were converted to text files using Bioworks. Theresulting text files were searched against a human ipi database usingthe automated search algorithm, X!Tandem.

Results and Discussion

As previously noted, the Western blot and coomassie-stained gel areshown in FIGS. 5A and 5B. In the Western blot, unique bands appear inthe lane containing the sample of interest that run at a molecularweight greater than the molecular weight of the biotinylated NKp30/mFc2(˜50 kDa). (See FIG. 5B.) This suggests that these bands arebiotinylated NKp30 crosslinked to binding partners on the surface of theK562 cells. In the corresponding coomassie-stained gel (see FIG. 5A),band 11 contains the proteins identified in the Western blot as NKp30conjugated to binding partners on the K562 cell surface. A list ofproteins identified from this section of the gel that were notidentified in the corresponding negative control bands (21, 31 and 41)can be found in Table 7. Analysis of the genomics database identifiedone of these proteins as hypothetical protein DKFZp686O24166. Thelocation of the three peptides identified by LC-MS/MS in the amino acidsequence of hypothetical protein DKFZP686I1167 can be found in FIG. 6.All spectra were also manually inspected to confirm the peptide/proteinidentifications made by X!Tandem.

TABLE 7 Unique proteins identified in gel band 11 Unique peptides ID'edProtein name by LC-MS/MS Natural cytotoxicity triggering 3 receptor 3Hypothetical protein 3 DKFZP686I21167 Plectin 6 6 Cation-independentmannose-6- 13 phosphate receptor precursor NKp30/Fc2 8

Conclusion

NKp30/mFc2 and hypothetical protein DKFZP686I21167 were identified onlyin the sample in which NKp30/mFc2 was allowed to interact with K562cells. These data support hypothetical protein DKFZP686I21167 as abinding partner to NKp30.

Example 5 Analysis of zB7H6 Sequence and Gene Structure andIdentification of zB7H6 as a B7 Family Member

Based on B7 family gene profiling, hypothetical protein DKFZP686I21167was identified as a member of the B7 family of cellular receptors. Thegene structure profile is Signal-2-IgV-2-IgC-2-TMD-0-LgEx. (See FIG. 7.)The extracellular region of this profile matches a B7 gene structuremodel, with includes characteristic exon patterns in which the firstexon encodes a leader sequence, the second exon encodes an IgV domainand the third exon encodes an IgC domain. Another characteristic featureof the B7 family gene structure is the phasing of the exons: in theregion corresponding to the extracellular domain, B7 family members showa conserved phasing of 2 between exons 1 to 4. (See id.) Based partly onthe identification of DKFZP686I21167 as a B7 family member, this proteinwas assigned the in-house designation zB7H6. zB7H6's cytoplasmic regionis homologous to Gag polyprotein with 44% identity, and it containspotential signaling motifs such as SaYtpL (ITIM), YqlQ (SH2), andPdaPilPvsP (SH3). (See FIG. 7.) Therefore, it may have other functionsin addition to triggering pNKp30.

A search of public EST databases identified at least 20 human ESTscorresponding to zB7H6, but no mouse EST or mRNAs. There are onlypredicted sequences for all the other species (e.g., mouse, rat, dog,cow). There is no similarity within the intracellular region betweenhuman and predicted peptides from other species except Apes.

Example 6 Human zB7H6 Expression Construct

cDNA clone CT#102296, corresponding to DKFZp686O24166 (designatedzB7H6), was purchased from the German Cancer Research Center,Heidelberg, Germany.

An expression plasmid containing a polynucleotide encoding thefull-length human zB7H6 (SEQ ID NO:2) was constructed via PCR,restriction digestion and ligation. A fragment of human zB7H6 cDNA wasisolated by PCR using CT#102296 as template, with flanking regions atthe 5′ and 3′ ends corresponding to the vector sequences flanking thehuman zB7H6 insertion point using primers zc58067 (SEQ ID NO:9) andzc58401 (SEQ ID NO:10)

The PCR reaction mixture was run on a 1% agarose gel and a bandcorresponding to the size of the insert is gel-extracted using aQIAquick™ Gel Extraction Kit (Qiagen, Valencia, Calif.). The resultingpurified PCR product was digested with EcoRI and XhoI for 2 hours at 37°C. and run on a 1% agarose gel for band purification as described above.Plasmid pZP-7NX was digested with EcoRI and XhoI for 2 hours at 37° C.and run on a 1% agarose gel for band purification as described above. 2μl of the PCR product and 1 μl of cut pZP-7NX were ligated in a totalvolume of 20 μl with 2 μl 10× ligation buffer, 14 ul of H₂0 and 1 ul ofT4 DNA Ligase (Promega, Madison, Wis.) for 2 hours at room temperature.1 ul of the ligation was electroporated into Electromax DH10B(Invitrogen, Carlsbad, Calif.) using a Gene Pulser II electroporator(BioRad, Hercules, Calif.) set at 25 uF, 300 ohms and 2100 volts. 100 μlof the transformation was plated on one LB AMP plates (LB broth(Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/L Ampicillin).

Individual colonies were grown overnight in a 2 ml LB 100 mg/LAmpicillin growth media and miniprepped using a plasmid mini kit(Qiagen, Valencia, Calif.) The minipreps were digested with BamHI andBglII and clones with the correct 1.152 kB insert were submitted for DNAsequencing. The correct construct was designated as pZP-7NX hzB7H6.

Example 7 Expression of Full-Length zB7H6 in P815 and BaF3 Cells: zB7H6Specifically Binds to NKp30 and is Able to Trigger NK Cell Activity

The zB7H6 clone that was verified sequence correct (pZP-7NX hzB7H6) wasreintroduced into electromax DH10B by electroporation and then scaled upto a 200 ml LB+amp overnight culture from which DNA was purified usingQiagen kit#12183. 40 μg of DNA was linearized by digestion with HindIIIand ethanol precipitated. This DNA was electroporated into P815 and BaF3cells using the following protocol. P815 cells were washed 2 times withOptimem serum free medium (Invitrogen, Carlsbad, Calif.) and resuspendedat 1×10⁷ cells/ml in Optimem. 800 μl of cells were transferred to thetube containing the linearized DNA from above and incubated for 15minutes at room temperature. The DNA/cell mix was transferred to a 4 mmelectroporation cuvette and shocked at 800 μF and 300 volts. After a 1minute incubation, cells were reshocked at 1180 μF and 300 volts. Cellswere incubated overnight at 37° C. before being selected in 1 mg/mlGeneticin (Invitrogen 1013-027) and clones were generated by plating bylimiting dilution at 0.3 cells/well. Randomly selected clones werescreened by FACS for binding to a soluble form of NKp30 (NKp30/VASP; SEQID NO:11). The highest selecting clone was put into a FACS bindingcompetition assay and a cytolytic assay.

A FACS binding competition assay was performed using BaF3 cellsresuspended at 3×10⁶/ml and aliquoted at 100 μl/sample for a final countof 300,000 cells/sample. 1 μl of whole mouse IgG (Jackson 015-000-003)was added per sample followed by addition of NKp30/mFc2-A647 labeledprobe at 2 μg/ml. In samples to include competition, unlabeled probe wasadded at 100-fold mass excess and the samples were incubated for 1 houron ice. Samples were washed one time with cold PBS and samples wereanalyzed for binding of soluble NKp30/mFc2-A647 on a FACSCalibur.

The results of the FACS binding competition assay are shown in FIGS. 8Aand 8B. Soluble NKp30/VASP-A647 bound to cells electroporated with thehzB7H6 expression vector, but not to control cells containing an emptyvector control. Staining with NKp30/VASP-A647 was not observed in thepresence of a 100-fold excess of unlabeled NKp30/VASP (see FIG. 8A), butwas observed in the presence of a 100-fold excess of unlabeledirrelevant VASP protein (see FIG. 8B).

A cytolytic assay was performed with NK-92 cells as effectors againstP815 targets. NK-92 cells were washed 1× with HBSSF (Hank's bufferedsaline (Ca, Mg Free)+5% FBS) and resuspended in HBSSF at 1.35×10⁶/ml (toachieve a 27:1 ratio). 150 μl of washed cells were plated in the top rowof a U-bottom 96-well plate and serially diluted (1:3) into HBSSF. P815target cells were washed 1× with HBSSF and labeled at 1×10⁶ cells/ml in10 μM calcein AM Molecular probes #C1430 (2.5 μl/ml of 4 mM stock inDMSO, 4 mM=4 mg/ml) for 60 minutes at 37 C. Labeled cells were washed 2×in HBSSF and 1×10⁶ cells were resuspended in 20 ml HBSSF (5000 cells/100μl). 100 μl of suspended target cells were added to diluted effectorsfor a total volume of 200 μl (effector:target ratios of 27:1, 9:1, 3:1,and 1:1). An activating anti-NKp30 monoclonal antibody was also added tosome sets of serially diluted wells at a concentration of 2 μg/ml.

Effector and target cells were spun at 500 rpm for 2 min, incubated at37° C. for 3 hours, spun at 1500 rpm for 5 min, and then 100 μlsupernatant transferred to a new flat bottom 96-well. Flat bottom platescontaining transferred supernatants were read on a fluoremeter for 1second at an excitation wavelength of 485 nm and emission wavelength of535 nm.

NK-92 cells did not lyse wild-type P815 cells or P815 cells transfectedwith two non-triggering control proteins (hIgSF1 (SEQ ID NO:14) andhB7H1 (SEQ ID NO:15)), while addition of an activating anti-NKp30monoclonal antibody triggered re-directed lysis. Transfection of eitherhCD86 (Azuma et al., Nature 366:76, 1993) or zB7H6 triggered directkilling of P815 cells.

These data demonstrate that zB7H6 specifically binds to NKp30 and isable to trigger cytolytic activity.

Example 8 Cloning and Construction of Human zB7H6/mFc2

An expression plasmid containing a polynucleotide encoding theextra-cellular domain of human zB7H6 and the mouse Fc2 portion wasconstructed via PCR amplification, restriction digestion and ligation. ADNA fragment of the extra-cellular domain of human zB7H6 was isolated byPCR using SEQ CT#102296 as template with flanking regions at the 5′ and3′ ends corresponding to the vector sequence and the mouse Fc2 sequenceflanking the human zB7H6 insertion point using primers zc50437 (SEQ IDNO:20) and zc50438 (SEQ ID NO:21).

The PCR reaction mixture was run on a 1% agarose gel and a bandcorresponding to the size of the insert is gel-extracted using aQIAquick™ Gel Extraction Kit (Qiagen, Valencia, Calif.). The initialplasmid used was pZMP21 as a base vector with the mouse Fc2 portionbuilt into it. Plasmid pZMP21 is a mammalian expression vectorcontaining an expression cassette having the MPSV promoter, multiplerestriction sites for insertion of coding sequences, a stop codon, an E.coli origin of replication; a mammalian selectable marker expressionunit comprising an SV40 promoter, enhancer and origin of replication, aDHFR gene, and the SV40 terminator; and URA3 and CEN-ARS sequencesrequired for selection and replication in S. cerevisiae. It wasconstructed from pZP9 (deposited at the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209, underAccession No. 98668) with the yeast genetic elements taken from pRS316(deposited at the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110-2209, under Accession No. 77145), aninternal ribosome entry site (IRES) element from poliovirus, and theextracellular domain of CD8 truncated at the C-terminal end of thetransmembrane domain. Plasmid hBTLA mFc2 pZMP21 was digested withEcoRl/BglII to cleave off human BTLA and used for ligation with the PCRinsert.

2 μl of the cut PCR product and 1 μl of cut pZMP21 were ligated in atotal volume of 20 ul with 2 ul 10× ligation buffer, 14 ul of H₂0 and 1ul of T4 DNA Ligase (Promega, Madison, Wis.) for 2 hours at roomtemperature. 1 ul of the ligation was electroporated into ElectromaxDH10B (Invitrogen, Carlsbad Calif.) using a Gene Pulser IIelectroporator (BioRad, Hercules, Calif.) set at 25 uF, 300 ohms and2100 volts. 100 μl of the transformation was plated on one LB AMP plate(LB broth (Lennox), 1.8% Bacto™ Agar (Difco), 100 mg/L Ampicillin). Thecolonies were screened by restriction digestion with EcoRI and KpnI,with clones showing the expected 1.596 kB insert being submitted for DNAsequencing. A sequence correct construct was designated ashB7H6mFc2pZMP21. The DNA sequence coding for hzB7H6/mFc2 is shown as SEQID NO:16; the amino acid sequence for hzB7H6/mFc2 is shown as SEQ IDNO:17.

Example 9 Cloning and Construction of zB7H6/VASP

Human vasodialator-activated phosphoprotein (VASP) is described byKühnel, et al. (Proc. Nat'l. Acad. Sci. USA 101: 17027, 2004). VASPnucleotide and amino acid sequences are provided as SEQ ID NOs: 3 and 4.Two overlapping oligonucleotides, which encoded both sense and antisensestrands of the tetramerization domain of human VASP protein, weresynthesized by solid phased synthesis using oligonucleotide zc50629 (SEQID NO:22) and oligonucleotide ZC 50630 (SEQ ID NO:23). Theseoligonucleotides were annealed at 55° C., and amplified by PCR with theolignucleotide primers zc50955 (SEQ ID NO:24) and zc50956 (SEQ IDNO:25).

The amplified DNA was fractionated on 1.5% agarose gel and then isolatedusing a Qiagen gel isolation kit according to manufacturer's protocol(Qiagen, Valiencia, Calif.). The isolated DNA was inserted into BglIIcleaved pzmp21 vector by yeast recombination. DNA sequencing confirmedthe expected sequence of the vector, which was designatedpzmp21VASP-His₆.

The extracellular domain of human zB7H6 was generated by PCRamplification from CT#102296 with oligos zc58284 (SEQ ID NO:26) andzc58419 (SEQ ID NO:27). The PCR reaction mixture was run on a 1% agarosegel and a band corresponding to the size of the insert is gel-extractedusing a QIAquick™ Gel Extraction Kit (Qiagen, Valencia, Calif.). Theresulting purified PCR product was digested with EcoRI and BglII for 2hours at 37° C. and run on a 1% agarose gel for band purification asdescribed above. The isolated fragment was inserted into EcoRI/BglIIcleaved pZMP21VASP-His₆ vector by ligation. 2 μl of the PCR product and1 μl of cut pZMP21VASP-His₆ were ligated in a total volume of 20 μl with2 μl 10× ligation buffer, 14 μl of H₂0 and 1 μl of T4 DNA Ligase(Promega, Madison, Wis.) for 2 hours at room temperature. 1 μl of theligation was electroporated into Electromax DH10B (Invitrogen, Carlsbad,Calif.) using a Gene Pulser II electroporator (BioRad, Hercules, Calif.)set at 25 μF, 300 ohms and 2100 volts. 100 μl of the transformation wasplated on one LB AMP plates (LB broth (Lennox), 1.8% Bacto™ Agar(Difco), 100 mg/L Ampicillin).

Individual colonies were grown overnight in a 2 ml LB AMP growth mediaand miniprepped using a plasmid mini kit (Qiagen, Valencia, Calif.) Theminipreps were digested with BamHI and BglII and clones with the correct1.152 kB insert were submitted for DNA sequencing. The correct constructwas designated as pZMP21 hzB7H6 VASP-His₆. The DNA sequence coding forhzB7H6/VASP-His₆ is shown as SEQ ID NO:18; the amino acid sequence forhzB7H6/VASP-His₆ is shown as SEQ ID NO:19.

Example 10 Stable Transfection and Expression of zB7H6/mFc2 in CHO Cells

Three sets of 50 μg of the hB7H6mFc2pZMP21 construct were each digestedwith 25 units of Pvu I at 37° C. for three hours and then wereprecipitated with IPA and spun down in a 1.5 mL microfuge tube. Thesupernatant was decanted off the pellet, and the pellet was washed with0.5 mL of 70% ethanol. The tube was spun in a microfuge for 15 minutesat 13,000 RPM and the supernatant was decanted off the pellet. Thepellet was then resuspended in 1 ml of ZF1 media in a sterileenvironment, allowed to incubate at 60° C. for 15 minutes, and wasallowed to cool to room temperature. 5E6 5×SA APFDXB11 CHO cells werespun down in each of three tubes and were resuspended using theDNA-media solution. The DNA/cell mixtures were placed in a 4 mm gapcuvette and electroporated using the following parameters: 950 μF, highcapacitance, and 300 V. The contents of the cuvettes were then removed,pooled, and diluted to 25 mLs with ZF1 media and placed in a 125 mLshake flask. The flask was placed in an incubator on a shaker at 37° C.,6% CO₂, and shaking at 120 RPM.

The cell line was subjected to nutrient selection followed by stepamplification to 200 nM methotrexate (MTX), and then to 500 nM MTX.Expression was confirmed by Western blot probed with anti-mouse IgG2aantibody and anti-mouse IgG H+L antibody, and the cell line wasscaled-up and protein purification followed.

Example 11 Stable Transfection and Expression of zB7H6/VASP in CHO Cells

Three sets of 50 μg of the pZMP21 hzB7H6 VASP-His₆ construct were eachdigested with 25 units of Pvu I at 37° C. for three hours and then wereprecipitated with IPA and spun down in a 1.5 mL microfuge tube. Thesupernatant was decanted off the pellet, and the pellet was washed with0.5 mL of 70% ethanol. The tube was spun in a microfuge for 15 minutesat 13,000 RPM and the supernatant was decanted off the pellet. Thepellet was then resuspended in 1 ml of ZF1 media in a sterileenvironment, allowed to incubate at 60° C. for 15 minutes, and wasallowed to cool to room temperature. 5E6 5×SA APFDXB11 CHO cells werespun down in each of three tubes and were resuspended using theDNA-media solution. The DNA/cell mixtures were placed in a 4 mm gapcuvette and electroporated using the following parameters: 950 μF, highcapacitance, and 300 V. The contents of the cuvettes were then removed,pooled, and diluted to 25 mLs with ZF1 media and placed in a 125 mLshake flask. The flask was placed in an incubator on a shaker at 37° C.,6% CO2, and shaking at 120 RPM.

The cell line was subjected to nutrient selection followed by stepamplification to 200 nM methotrexate (MTX), and then to 500 nM MTX.Expression was confirmed by Western blot probed with anti-6-Hisantibody, and the cell line was scaled-up and protein purificationfollowed.

Example 12 zB7H6 Triggers Cytolytic Activity in Human Primary NK Cells

A cytolytic assay was performed with human primary NK cells as effectorsagainst P815 targets. NK cells were purified from human peripheral bloodusing negative selection with magnetic bead labeling Miltenyi#130-092-657. These purified NK cells were cultured overnight inRPMI/10% FBS supplemented with 10 ng/ml of human IL-2 (R&D #202-IL-010).The NK cells were then washed 1× with HBSSF (Hank's buffered saline (Ca,Mg Free)+5% FBS) and resuspended in HBSSF at 1.35×106/ml (to achieve a27:1 ratio). 150 μl of washed cells were plated in the top row of aU-bottom 96-well plate and serially diluted (1:3) into HBSSF. P815target cells were washed 1× with HBSSF and labeled at 1×10⁶ cells/ml in10 μM calcein AM Molecular probes #C1430 (2.5 μl/ml of 4 mM stock inDMSO, 4 mM=4 mg/ml) for 60 minutes at 37° C. Labeled cells were washed2× in HBSSF and 1×10⁶ cells were resuspended in 20 ml HBSSF (5000cells/100 μl). 100 μl of suspended target cells were added to dilutedeffectors for a total volume of 200 μl (effector:target ratios of 27:1,9:1, 3:1, and 1:1). An activating anti-NKp30 monoclonal antibody wasalso added to some sets of serially diluted wells at a concentration of2 μg/ml. A soluble mFc version of NKp30 was added at 2 μg/ml to somesets of serially diluted wells and an unrelated protein, HHLA2/mFc2 wasadded to a different set at the same concentration.

Effector and target cells were spun at 500 rpm for 2 min, incubated at37° C. for 3 hours, spun at 1500 rpm for 5 min, and then 100 μl ofsupernatant was transferred to a new flat bottom 96-well. Flat bottomplates containing transferred supernatants were read on a fluoremeterfor 1 second at an excitation wavelength of 485 nm and emissionwavelength of 535 nm.

NK cells lysed wild-type P815 cells at low levels but P815 cellstransfected with zB7H6 were lysed at levels approximating re-directedkilling triggered by an activating anti-NKp30 monoclonal antibody.Soluble NKp30 inhibited lysis of zB7H6 transfected P815 to approximatelybackground levels, but addition of HHLA2/mFc2 had no effect.

Example 13 Generation of Mouse Anti-zB7H6 Polyclonal AntibodyImmunizations

Five 3 month old female BALB/c mice (Charles River Laboratories,Wilmington, Mass.) were immunized with human zB7H6. The mice wereinitially immunized by subcutaneous injection with ˜50 μg of purified,recombinant human zB7H6 (ZGI produced in CHO DXB 11 5SA, Lot # A1980F)fused with VASP, 6His, and BSA conjugated (SJAS 9 Aug. 2007) incombination with Emulsigen®-P adjuvant (MVP Laboratories INC, Omaha,Nebr.) as per manufacturer's instructions. Following the initialimmunization each of the mice received an additional 50 μg of humanzB7H6 in Emulsigen®-P adjuvant via the subcutaneous route every twoweeks over a nine week period. Seven days after the third and fourthimmunizations the mice were bled via the retro orbital plexus and theserum was separated from the blood for analysis of its ability to bindto human zB7H6.

Direct Assay

The ability of anti-human zB7H6 antibodies in the anti-sera to bind tohuman zB7H6 (lot# A1980F) was assessed using a direct style ELISA assay.In this assay, wells of 96-well polystyrene ELISA plates were firstcoated with 100 μL/well of human zB7H6 (lot # A1980F) at a concentrationof 1 μg/mL in Coating Buffer (0.1M Na2CO3, pH 9.6). Plates wereincubated overnight at 4° C. after which unbound receptor was aspiratedand the plates washed twice with 300 μL/well of Wash Buffer (PBS-Tweendefined as 0.137M NaCl, 0.0022M KCl, 0.0067M Na2HPO4, 0.0020M KH2PO4,0.05% v/w polysorbate 20, pH 7.2). Wells were blocked with 200 μL/wellof Blocking Buffer (PBS-Tween plus 1% w/v bovine serum albumin (BSA))for 1 hour, after which the plates were washed twice with Wash Buffer.Serial 10-fold dilutions (in 1% BSA in PBS-Tween) of the sera wereprepared beginning with an initial dilution of 1:100 and ranged to1:100,000. Normal mouse sera served as a control. Duplicate samples ofeach dilution were then transferred to the assay plate, 100 μL/well inorder to bind human zB7H6. Following a 1 hour incubation at roomtemperature, the wells were aspirated and the plates washed twice asdescribed above. Horseradish peroxidase labeled Goat anti Mouse Kappaantibody (SouthernBiotech, Birmingham, Ala.) at a dilution of 1:5,000was then added to each well, 100 μL/well, and the plates incubated at RTfor 1 hour. After removal of unbound HRP conjugated antibody, the plateswere washed twice, 100 μL/well of tetra methyl benzidine (TMB) (BioFXLaboratories, Owings Mills, Md.) added to each well and the platesincubated for 2.5 minutes at RT. Color development was stopped by theaddition of 100 μL/well of TMB Stop Reagent (BioFX Laboratories, OwingsMills, Md.) and the absorbance values of the wells read on a MolecularDevices Spectra MAX 340 instrument at 450 nm.

Immune sera from all mice showed a strong anti-VASP and anti-zB7H6response. Sera was pooled and anti-zB7H6 antibody was purified asdescribed below.

Purification of zB7H6 Polyclonal Antibodies

Serum from mice challenged with zB7H6C(VASP)H6 was pooled, diluted 1:1(v/v) with 35 mM NaPO4, 120 mM NaCl, pH 7.2 and 0.2 μm sterile filteredprior to loading (via batch method) onto CNBr-activated Sepharose™ 4B(GE Healthcare, Piscataway, N.J.) coupled with zB7H6 (mFc2). Prior toloading the diluted serum, the CNBr-activated Sepharose™ 4B resin waspre-equilibrated with, 20 column volumes (approximately 50 ml) of 35 mMNaPO₄, 120 mM NaCl, pH 7.2 The ratio of diluted serum to coupled resinwas 2.8:1 (v/v).

The chromatography process was performed at both 5° C. and ambient roomtemperature. Specifically, the loading (capture step) of the dilutedserum onto the zB7H6 (mFc2) coupled CNBr-activated Sepharose™ 4B resinwas performed using a rocking platform at 5° C. The wash step andsubsequent elution step were performed at ambient room temperature(approximately 22° C.) after the serum/resin slurry was poured into anempty glass Econo-Column (Bio-Rad, Hercules, Calif.). The column waswashed (via gravity flow) with 15 column volumes (approximately 37.5 ml)of 35 mM NaPO₄, 120 mM NaCl, pH 7.2. Bound antibody was then pH eluted(via gravity flow) with 100 mM glycine, pH 2.7. 0.5 ml fractions werecollected and immediately neutralized with 0.05 ml 2.0M Tris-HCl, pH8.0. Fractions were collected and pooled based on A280 readings from aNanoprop (Thermo Scientific, Fremont, Calif.). The retained flow-throughwas then reapplied to the zB7H6 (mFc2) coupled CNBr-activated Sepharose™4B resin after column regeneration/equilibration. This batch/elute cyclewas repeated two times.

The pooled fractions of the corresponding purifications were pooled andthen desalted (buffer-exchanged) against 35 mM NaPO₄, 120 mM NaCl, pH7.2 using pre-packed Sephadex™ G-25 Superfine columns, HiTrap™ columns(GE Healthcare, Piscataway, N.J.). 0.5 ml fractions were collected. Thepooling of these fractions was determined by the A280 reading on theAKTA Explorer. Pooled, desalted, fractions were then 0.22 μm sterilefiltered prior to aliquoting and storage at −80° C.

Example 14 Validation of Mouse Anti-zB7H6 Polyclonal Antibody Activityand Specificity

Mouse anti-zB7H6 affinity purified polyclonal antibody was conjugatedwith Alexa-647 fluorescent marker using an Alexafluor-A647 antibodylabeling kit (Invitrogen A30009) following the manufacturer'sinstructions. 150,000 cells/sample wild-type or zB7H6-transfected P815cells were probed with anti-zB7H6-A647 at 1 μg/ml with or withoutunlabeled competitors at 100-fold mass excess. Cells were incubated for1 hour on ice, washed once with 2 ml of ice cold PBS and then read byflow cytometry on a FACSCalibur instrument. Binding was recorded as meanfluorescent intensity (MFI). Results of this study showed thatanti-zB7H6-A647 antibody bound to zB7H6-transfected P815 cells (MFI600), but not to wild-type (untransfected) P815 cells (MFI≈25). Thisbinding was competable with a 100-fold mass excess of unlabeledanti-zB7H6 (MFI≈40), but not with a 100-fold mass excess of an isotypecontrol antibody (MFI≈500).

Mouse anti-zB7H6 polyclonal antibody was also used in a competitionbinding assay of NKp30/mFc2-biotin binding to P815 transfectants.150,000 wild-type or zB7H6-transfected P815 cells were probed withNKp30/mFc2-biotin at 1 μg/ml in 100 μl PBS/2% FBS. Unlabeled anti-zB7H6polyclonal antibody or other control antibodies or soluble receptorswere added at 100-fold mass excess. Cells were stained for 1 hour onice, washed once with 2 ml of ice cold PBS, and then stained withstreptavidin-PE at 1 μg/ml (BD:554061) for 15 min. on ice. Cells wereagain washed with cold PBS before being read by flow cytometry on aFACSCalibur instrument. Binding was recorded as mean fluorescentintensity (MFI). Results of this study showed that NKp30/mFc2-biotinbound to zB7H6-transfected cells (MFI≈825), but not to wild-type P815cells (MFI<15). Binding of labeled NKp30/mFc2 was competable by bothunlabeled anti-zB7H6 antibody and NKp30/mFc2 (MFI≈25), but not with anisotype control antibody (MFI≈775).

Example 15 Inhibition of NK-92 Cytolytic Activity Against K562 and P815zB7H6 Targets with Soluble Proteins

A cytolytic assay was performed with NK-92 cells as effectors againstK562 and P815 zB7H6 targets.

NK-92 cells were washed 1× with HBSSF (Hank's buffered saline (Ca, MgFree)+5% FBS) and resuspended in HBSSF at 1.35×10⁶/ml (to achieve a 27:1ratio). 150 μl of washed cells were plated in the top row of a U-bottom96-well plate and serially diluted (1:3) into HBSSF.

K562 and P815 zB7H6 target cells were washed 1× with HBSSF and labeledat 1×10⁶ cells/ml in 10 μM calcein AM Molecular probes #C1430 (2.5 μl/mlof 4 mM stock in DMSO, 4 mM=4 mg/ml) for 60 minutes at 37° C. Labeledcells were washed 2× in HBSSF and 1×10⁶ cells were resuspended in 20 mlHBSSF (5000 cells/100 μl). 100 μl of suspended target cells were addedto diluted effectors for a total volume of 200 μl. A soluble form ofNKp30 (NKp30VASP tetrameric receptor), a VASP control (B7H3/VASP; SEQ IDNO:[28]), an anti-zB7H6 polyclonal antibody (E10607), or an irrelevantcontrol antibody was also added to some sets of serially diluted wellsat a concentration of 5 μg/ml.

Effector and target cells were spun at 500 rpm for 2 minutes, incubatedat 37° C. for 3 hours, spun at 1500 rpm for 5 minutes, and then 100 μlsupernatant transferred to a new flat bottom 96-well. Flat bottom platescontaining transferred supernatants were read on a fluoremeter for 1second at an excitation wavelength of 485 nm and emission wavelength of535 nm.

As shown in FIG. 10, soluble NKp30/VASP and anti-zB7H6 polyclonalantibody inhibited the cytolytic activity of NK-92 cells against K562and P815 zB7H6 targets at a 9:1 effector to target ratio. (See FIG. 10.)Inhibition was also seen at target to effector ratios of 27:1 and 3:1.VASP and irrelevant antibody controls had no effect. These data suggestthat the ability of NK-92 to lyse K562 and P815 zB7H6 targets isNKp30-mediated and is further dependent on zB7H6.

Example 16 Soluble NKp30 Specifically Binds K562, P815 zB7H6 and 293FCells

K562, P815 zB7H6 and 293F cells were probed by FACS with a biotinylatedsoluble form of NKp30 (Kp30/mFc2), containing the extracellular domainof NKp30 and a murine Fc fragment. Cells were resuspended in PBS/2% FBSat a concentration of 1.5×10⁶ cells/ml (150,000 cells/sample). 100 μlsamples were aliquoted with 100 μg/ml of whole human IgG (Jackson#009-000-003) included for Fc receptor blocking. NKp30/mFc2-biotin probewas added at a concentration of 2 μg/ml and 100-fold mass excess of aVASP protein (NKp30VASP or human zB7H6/VASP) or a control VASP protein(B7H3/VASP). Cells were incubated for 1 hour on ice and washed with 2 mlcold PBS. Washed cells were resuspended in 100 μl of PBS/2% FBS withstreptavidin-PE (BD:554061) at 1 μg/ml and incubated for 15 minutes onice. Cells were then washed with 1 ml cold PBS, resuspended in 250 μl ofPBS, and analyzed for PE staining on a FACSCalibur.

As shown in FIG. 11, NKp30/mFc2-biotin bound to K562, 293F and P815zB7H6 cells (“No Competition”). This binding was competable withNKp30/VASP and zB7H6/VASP, but not with control VASP protein (B7H3/VASP)demonstrating that the binding of NKp30/mFc2 to K562, P815 zB7H6 and293F cells was specific. Little or no binding was observed for MCF-7,Aspc-1, A549, and HL-60 tumor cell lines.

Example 17 Anti-zB7H6 Specifically Binds K562, P815 zB7H6 and 293F Cells

K562, P815, P815 zB7H6 and 293F cells were probed with an A647conjugated form of anti-zB7H6 mouse polyclonal antibody (E10607). Cellswere resuspended in PBS/2% FBS at a concentration of 1.5×10⁶ cells/ml(150,000 cells/sample). 100 μl samples were aliquoted with 100 μg/ml ofwhole human IgG (Jackson #009-000-003) included to block Fc receptors.Anti-zB7H6-A647 antibody was added at a concentration of 2 μg/ml and100-fold mass excess of a VASP protein (zB7H6/VASP or a control VASPprotein (B7H3/VASP)). Cells were incubated for 1 hour on ice and washedwith 2 ml cold PBS. Cells were then resuspended in 250 μl of PBS andanalyzed for APC staining on a FACSCalibur.

As shown in FIG. 12, anti-zB7H6 bound to K562, P815 zB7H6 and 293F cellsbut not to untransfected P815 cells (“No Competition”). This binding wascompetable with zB7H6/VASP, but not with control VASP protein(B7H3/VASP), demonstrating that the binding of anti-zB7H6-A647 to K562,P815 zB7H6 and 293F cells was specific. Little or no binding wasobserved for MCF-7, Aspc-1, A549, and HL-60 tumor cell lines. Thesedata, taken together with the NKp30/mFc2-biotin binding data, show thecorrespondence of NKp30/mFc-biotin binding with zB7H6 expression.

Example 18 Quantitative Real Time PCR Analysis of Normal Human Tissues

Quantitative real-time polymerase chain reaction (qRT-PCR) was used toassay zB7H6 mRNA message levels in normal human tissues. zB7H6 primerand probe were purchased from ABI using their proprietary software thatgenerates primers with FAM reporter dye designed to span exon/intronboundaries to avoid amplification from genomic DNA. This primer(ABI:Hs02340611_m1) was used in a validation experiment in combinationwith a primer for the housekeeping gene HPRT1 (ABI:4333768-0712016) on293F cDNA in a 5 log dilution series starting at 100 ng. A plot of theLog of 293F cDNA concentration versus delta cycle threshold (deltaCt)gave a statistically fitted line with the formula Y=−0.02571x+3.504indicating that the efficiencies of zB7H6 primer and probe set matchedthat of HPRT1 making Log₂ Ct calculations valid (a passing validationexperiment is defined as the absolute value of the slope of deltaCt vs.log input cDNA <0.1). A no reverse transcriptase (−RT) control wasperformed for each of the concentrations in the 293F dilution series toverify the absence of amplification from genomic DNA. A Normal tissueqPCR array was purchased from Origene (Origene HMRT102). 1st strandcDNAs from poly-A RNA in this array were normalized for GAPDH by themanufacturer. Lyophilized samples were resuspended in 30 μl DiH₂0 and13.5 μl was split into each of two reactions, one for HPRT1 and one forzB7H6 RT-PCR. Primers were used at 900 nM and probe at 250 nM in 10 μlreactions run in triplicate on an ABI 7900HT RT-PCR instrument. Noamplification with the zB7H6 primers in any of the 48 normal tissuessamples was observed despite amplification of the HPRT1 housekeepinggene amplifying in all samples. Additionally, a 293F positive controlcDNA gave zB7H6 amplification, indicating that the qRT-PCR reaction wasworking properly.

Example 19 Quantitative Real-Time PCR Analysis of Tumor Cell Lines

qRT-PCR was also used to evaluate zB7H6 mRNA from a panel of tumor celllines of various origin. Total RNA was generated from cells using RNeasyMidi columns (Qiagen 75142) following the manufacturer's instructions.First strand cDNA was synthesized by reverse transcription of 1 μg ofRNA using Invitrogen Superscript III Kit (Invitrogen 11752-250)following the manufacturer's instructions. The same primer and probesets as described in Example 18, supra, were used to analyze 19.3 ng of1st strand cDNA from tumor lines. Daudi cells, which were observed tohave low binding levels of NKp30/mFc2 and anti-zB7H6, gave a Log₂ Ctaverage value of 0.079 from 3 different reactions run in triplicate onthree different days; therefore, 0.07 was used as a threshold to definezB7H6 positivity in the qRT-PCR assay. 23 of the 118 cell lines assayedwere found to express zB7H6 message. Tumor cell lines expressing zB7H6are listed in Table 8, below.

TABLE 8 zB7H6 positive tumor cell-lines Cell-line Source 2{circumflexover ( )}Ct NCI-H716 Colon 0.152 hct15 Colon 0.219 hct116 Colon 0.070ht29 Colon 0.160 HEP3B2.1.7 Liver 0.071 HuH7 Liver 0.075 C3a Liver 0.249hepg2 Liver 0.146 Hela Cervix 0.097 SHP-77 Lung 0.076 NCI-H441 Lung0.152 BxPC3 Pancreas 0.983 Aspc-1 Pancreas 0.074 LN-CAP-FGC Prostate0.095 HL-60 prohemocytic leukemia 0.080 GRANTA519 B-cell lymphoma 0.115DOHH2 B-cell lymphoma 0.088 U-937 Monocytic lymphoma 0.184 HEL92.1.7Erythroleukemia 0.098 Daudi Burkitt's lymphoma 0.079 K562 chronicmyelogenous leukemia 0.080 293F 0.091 MV-4-11 0.130

Example 20 BxPC3 Pancreatic Carcinoma Model for Evaluating Efficacy ofan Anti-zB7H6 Antibody or Antibody-Drug Conjugate Against Tumor Growth

To test if an anti-zB7H6 antibody or antibody-drug conjugate hasactivity on tumor growth in mice, groups of mice are injected s.c withthe BxPC3 pancreatic tumor on Day 0. Once tumors grow to 150-200 mm³,groups of mice (n=10/gp) mice are then injected with 1 mg/Kg to 30 mg/Kgcontrol reagent, anti-zB7H6 antibody, or anti-zB7H6 antibody-drugconjugate 1×-3×/week for 3 weeks. Tumor volume is monitored 3×/week for5 weeks. Significantly smaller tumors in mice injected with a ananti-zB7H6 antibody or antibody-drug conjugate, as compared to miceinjected with control reagent, indicates efficacy of the antagonist forinhibition of tumor growth.

Study design: Eight to ten-week old female C.B-17 SCID mice (CharlesRiver Laboratories) are injected s.c. on the right flank with 2×10⁶BxPC-3 cells on Day 0, Starting with a tumor size of 150-200 mm³, groupsof mice (n=10/group) are injected i.p. with 1 mg/Kg to 30 mg/Kg controlreagent, anti-zB7H6 antibody, or anti-zB7H6 antibody-drug conjugate1×-3×/week for 3 weeks. Tumor growth is monitored 3×/week for 5 weeksusing caliper measurements. Tumor volume is calculated using the formula½*(B)²*L (mm³).

Example 21 Inhibition of Human Hepatocellular Carcinoma Cell Growth InVivo Using Anti-zB7H6 Antibody or Antibody-Drug Conjugate

To evaluate anti-tumor activity of an anti-zB7H6 antibody orantibody-drug conjugate against human hepatocellular carcinoma cells invivo, groups of BALB/c nude mice are injected with either HuH7 or C3Ahepatocellular carcinoma cells on Day 0. Groups (n=10/group) of tumorbearing mice receive 5-75 μg of anti-zB7H6 antibody or antibody-drugconjugate by i.p. or peritumoral injection every other day (EOD) fromDays 5-33. Tumor volume is monitored 3×/week for 6 weeks. Inhibition oftumor growth by anti-zB7H6 antibody or antibody-drug conjugate indicatesthat the respective protein has inhibitory effects on humanheptocellular carcinoma in vivo.

Study design: Eight-week old female BALB/c nude mice (Charles RiverLaboratories) are injected s.c. on the right flank with 6×10⁶ HuH7 orC3A cells on Day 0. Groups of mice (n=10/group) are injected i.p. orperitumorally with 5 μg-75 μg of an anti-zB7H6 antibody or anti-zB7H6antibody-drug conjugate from days 5-33. Injections are given in a totalvolume of 200 μl. Tumor growth is monitored 3×/week for 6 weeks usingcaliper measurements. Tumor volume was calculated using the formula½*(B)²*L (mm³).

Example 22 Inhibition of Human Prostate Carcinoma Cell Growth In VivoUsing Anti-zB7H6 Antibody or Antibody-Drug Conjugate

To evaluate anti-tumor activity of an anti-zB7H6 antibody orantibody-drug conjugate against human prostate carcinoma cells in vivo,groups of BALB/c nude mice are injected with either PC-3 or DU-145prostate carcinoma cells on Day 0. Groups (n=10/group) of tumor bearingmice receive 5-75 μg of anti-zB7H6 antibody or anti-zB7H6 antibody-drugconjugate by i.p. or peritumoral injection every other day (EOD) fromDays 5-33. Tumor volume is monitored 3×/week for 6 weeks. Inhibition oftumor growth (volume or weight) by an anti-zB7H6 antibody orantibody-drug conjugate indicates that the respective protein hasinhibitory effects on human prostate carcinoma in vivo.

Study design: Eight-week old female BALB/c nude mice (Charles RiverLaboratories) are injected s.c. on the right flank or orthotopically inthe prostate lobe with 10×10⁶ PC-3 or 6×10⁶ DU-145 cells on Day 0.Groups of mice (n=10/group) are injected i.p. or peritumorally (s.cmodel only) with 5-75 μg of anti-zB7H6 antibody or anti-zB7H6antibody-drug conjugate from days 5-33. Injections are given in a totalvolume of 200 μl. For s.c tumors, tumor growth is monitored 3×/week for6 weeks using caliper measurements. Tumor volume is calculated using theformula ½*(B)²*L (mm³). For orthotopic tumors, mice are terminated atthe end of the study and tumor weighed to enable tumor load assessment.

Example 23 Inhibition of Human Colon Carcinoma Cells In Vivo UsingAnti-zB7H6 Antibody or Antibody-Drug Conjugate

To evaluate anti-tumor activity of an anti-zB7H6 antibody orantibody-drug conjugate against human colon carcinoma cells in vivo,groups of BALB/c nude mice are injected with either DLD-1 or HCT-116colon carcinoma cells on Day 0. Groups (n=10/group) of tumor bearingmice receive 5-75 μg of anti-zB7H6 antibody or anti-zB7H6 antibody-drugconjugate by i.p. or peritumoral injection every other day (EOD) fromDays 5-33. Tumor volume is monitored 3×/week for 6 weeks. Inhibition oftumor growth (volume or weight) by anti-zB7H6 antibody or antibody-drugconjugate suggests that the respective protein has inhibitory effects onhuman colon carcinoma in vivo.

Study design: Eight-week old female BALB/c nude mice (Charles RiverLaboratories) are injected s.c. on the right flank or orthotopically inthe colonic wall with 6×10⁶ DLD-1 or HCT-116 cells on Day 0. Groups ofmice (n=10/group) are injected i.p. or peritumorally (for s.c modelonly) with 5-75 μg of anti-zB7H6 antibody or anti-zB7H6 antibody-drugconjugate from days 5-33. Injections are given in a total volume of 200μl. For s.c tumors, tumor growth is monitored 3×/week for 6 weeks usingcaliper measurements. Tumor volume is calculated using the formula½*(B)²*L (mm³). For orthotopic tumors, mice are terminated at the end ofthe study and tumor weighed to enable tumor load assessment.

Example 24 Inhibition of Human Pancreatic Carcinoma Cells In Vivo UsingAnti-zB7H6 Antibody or Antibody-Drug Conjugate

To evaluate anti-tumor activity of an anti-zB7H6 antibody orantibody-drug conjugate against human pancreatic carcinoma cells invivo, groups of BALB/c nude mice are injected with either BxPC-3 orHPAF-II pancreatic carcinoma cells on Day 0. Groups (n=10/group) oftumor bearing mice receive 5-75 μg of anti-zB7H6 antibody or anti-zB7H6antibody-drug conjugate by i.p. or peritumoral injection every other day(EOD) from Days 5-33. Tumor volume is monitored 3×/week for 6 weeks.Inhibition of tumor growth (volume or weight) by anti-zB7H6 antibody orantibody-drug conjugate suggests that the respective protein hasinhibitory effects on human pancreatic carcinoma in vivo.

Study design: Eight-week old female BALB/c nude mice (Charles RiverLaboratories) are injected s.c. on the right flank or orthotopically inthe pancreatic lobe with 6×10⁶ BxPC-3 or HPAF-II cells on Day 0. Groupsof mice (n=10/group) are injected i.p. or peritumorally (for s.c modelonly) with 5-75 μg of anti-zB7H6 antibody or anti-zB7H6 antibody-drugconjugate from days 5-33. Injections are given in a total volume of 200μl. For s.c tumors, tumor growth is monitored 3×/week for 6 weeks usingcaliper measurements. Tumor volume was calculated using the formula½*(B)²*L (mm³). For orthotopic tumors, mice are terminated at the end ofthe study and tumor weighed to enable tumor load assessment.

Example 25 Inhibition of B-Cell Lymphoma In Vivo Using Anti-zB7H6Antibody or Antibody-Drug Conjugate

Human B-lymphoma cell lines are maintained in vitro by passage in growthmedium. The cells are washed thoroughly in PBS to remove culturecomponents.

SCID Mice are injected with (typically) 1×10⁶ human lymphoma cells viathe tail vein in a 100 microliter volume. The optimal number of cellinjected is determined empirically in a pilot study to yield tumor takeconsistently with desired kinetics. Anti-zB7H6 antibody or antibody-drugconjugate treatment is begun the next day by either subcutaneousimplantation of an ALZET® osmotic mini-pump (ALZET, Cupertino, Calif.)or by daily i.p. injection of anti-zB7H6 antibody or antibody-drugconjugate or vehicle. Mice are monitored for survival and significantmorbidity. Mice that lose greater than 20% of their initial body weightare sacrificed, as well as mice that exhibit substantial morbidity suchas hind limb paralysis. Depending on the lymphoma cell line employed,the untreated mice typically die in 3 to 6 weeks. For B cell lymphomasthat secrete IgG or IgM, the disease progression can also be monitoredby weekly blood sampling and measuring serum human immunoglobulin levelsby ELISA.

Anti-zB7H6 Antibody or Antibody-Drug Conjugate Dose Response/IM-9 Model

Mice are injected with 1×10⁶ IM-9 cells, and 28 day osmotic mini pumpsimplanted the following day. The pumps are loaded with the followingconcentrations of zB7H6 antibody or antibody-drug conjugate to deliver:0, 0.12, 1.2, or 12 micrograms per day with 8 mice per dose group.Increased protection of mice from the tumor cell line with increaseddose of antibody or antibody-drug conjugate indicates that the effectsof the anti-zB7H6 antibody or antibody-drug conjugate are dosedependent. Surviving mice at the end of the experiment have no signs ofdisease and no detectable human IgG in their serum.

These data demonstrate that the efficacy of anti-zB7H6 antibody oranti-zB7H6 antibody-drug conjugate in SCID mouse lymphoma modelscorrelates with the ability to inhibit the growth of the lymphoma celllines in vivo.

Example 26 Inhibition of B-Cell Derived Tumors In Vivo Using Anti-zB7H6Antibody or Antibody-Drug Conjugate

Administration of anti-zB7H6 antibody or anti-zB7H6 antibody-drugconjugate by constant infusion via mini-osmotic pumps results in steadystate serum concentrations proportional to the concentration of theantibody or antibody-drug conjugate contained in the pump. 0.22 ml ofanti-zB7H6 antibody or antibody-drug conjugate contained in phosphatebuffered saline (pH 6.0) at a concentration of 2 mg/ml or 0.2 mg/ml isloaded under sterile conditions into Alzet mini-osmotic pumps (model2004; Alza corporation Palo Alto, Calif.). Pumps are implantedsubcutaneously in mice through a 1 cm incision in the dorsal skin, andthe skin is closed with sterile wound closures. These pumps are designedto deliver their contents at a rate of 0.25 μl per hour over a period of28 days. This method of administration results in significant increasein survival in mice injected with tumor cells (below).

Effect of Anti-zB7H6 Antibody or Antibody Drug Conjugate on B-CellDerived Tumors In Vivo

The effects of anti-zB7H6 antibody or antibody-drug conjugate are testedin vivo using a mouse tumor xenograft model described herein. Thexenograft model to be tested is human lymphoblastoid cell line IM-9(ATCC No. CRL159). C.B-17 SCID mice (female C.B-17/IcrHsd-scid; Harlan,Indianapolis, Ind.) are divided into 4 groups. On day 0, IM-9 cells(ATCC No. CRL159) are harvested from culture and injected intravenously,via the tail vein, to all mice (about 1,000,000 cells per mouse). On day1, mini-osmotic pumps containing test article or control article areimplanted subcutaneously in the mice. Mice in groups 1-3 (n=9 per group)are delivered anti-zB7H6 antibody or antibody-drug conjugate: group 1contains 2.0 mg/mL of antibody or antibody-drug conjugate and isdelivered 12 μg per day; group 2 contains 0.20 mg/mL and is delivered1.2 μg per day; group 3 contained 0.02 mg/mL and is delivered 0.12 μgper day. Mice in group 4 (n=9) are a control and are treated withvehicle (PBS pH 6.0).

Increased survival of treatment groups (e.g., either 12 μg/day or 1.2μg/day) compared to vehicle treated mice shows that anti-zB7H6 antibodyor antibody-drug conjugate reduces the effects of the B-cell tumor cellsin vivo.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims. All publications, patents, andpatent applications cited herein are hereby incorporated by reference intheir entireties for all purposes.

1. An isolated, soluble polypeptide comprising a polypeptide segmentthat has at least 90% sequence identity with the amino acid sequence setforth in residues 25-266 of SEQ ID NO:2, wherein said polypeptide iscapable of specifically binding to human NKp30.
 2. The polypeptide ofclaim 1, wherein the polypeptide segment consists of the amino acidsequence set forth in residues 25-266 of SEQ ID NO:2.
 3. The polypeptideof claim 1, which is a soluble fusion protein.
 4. The polypeptide ofclaim 3, wherein the polypeptide segment consists of the amino acidsequence set forth in residues 25-266 of SEQ ID NO:2.
 5. The polypeptideof claim 3, further comprising an immunoglobulin heavy chain constantregion.
 6. The polypeptide of claim 5, wherein the immunoglobulin heavychain constant region is an F_(c) fragment.
 7. The polypeptide of claim5, wherein the immunoglobulin heavy chain constant region is an isotypeselected from the group consisting of an IgG, IgM, IgE, IgA, and IgD. 8.The polypeptide of claim 7, wherein the IgG isotype is IgG₁, IgG₂, IgG₃,or IgG₄.
 9. The polypeptide of claim 3, further comprising a VASPdomain.
 10. An isolated polynucleotide comprising: a polynucleotidesegment encoding the polypeptide of claim
 1. 11. A vector comprising thepolynucleotide of claim
 10. 12. An expression vector comprising thefollowing operably linked elements: a transcription initiation region; aDNA segment encoding the polypeptide of claim 1; and a transcriptiontermination region.
 13. A host cell comprising the expression vector ofclaim
 12. 14. A method of producing a soluble zB7H6 polypeptide, themethod comprising: culturing the host cell of claim 13 under conditionsin which the polypeptide is expressed; and recovering the expressedpolypeptide.
 15. An isolated antibody that specifically binds to apolypeptide segment consisting of the amino acid sequence set forth inresidues 25-266 of SEQ ID NO:2.
 16. The isolated antibody of claim 15,wherein the antibody inhibits the interaction of zB7H6 with human NKp30.17. The antibody of claim 15, wherein the antibody is a monoclonalantibody.
 18. The antibody of claim 17, wherein the antibody is a humanor humanized monoclonal antibody.
 19. The antibody of claim 17, whereinthe antibody is a single chain antibody.
 20. The antibody of claim 16,wherein the antibody comprises an Fc region having at least one of ADCCactivity and CDC activity.
 21. The antibody of claim 20, wherein the Fcregion is a single chain Fc (scFc).
 22. A pharmaceutical compositioncomprising: the antibody of claim 16; and a pharmaceutically acceptablecarrier.
 23. A method for enhancing human natural killer (NK) cellactivity, the method comprising: contacting a human NK cell with a cellexpressing a recombinant, membrane-bound zB7H6 polypeptide comprising apolypeptide segment that has at least 90% sequence identity with theamino acid sequence set forth in residues 25-266 of SEQ ID NO:2, whereinsaid zB7H6 polypeptide is capable of specifically binding to humanNKp30.
 24. The method of claim 23, wherein the polypeptide segmentconsists of the amino acid sequence set forth in residues 25-266 of SEQID NO:2.
 25. A method for decreasing human natural killer (NK) cellactivity against a cell expressing zB7H6, the method comprising:contacting a cell expressing functional zB7H6, in the presence of ahuman NK cell, with an effective amount of an antibody that specificallybinds to a polypeptide segment consisting of the amino acid sequence setforth in residues 25-266 of SEQ ID NO:2, wherein said antibody inhibitsthe interaction of zB7H6 with human NKp30.
 26. A method for treatingbone marrow cell (BMC) allograft rejection in a subject, the methodcomprising: administering to the subject, in an amount effective toinhibit NK cell activity and thereby treat the acute BMC allograftrejection, an antibody that specifically binds to a polypeptide segmentconsisting of the amino acid sequence set forth in residues 25-266 ofSEQ ID NO:2, wherein said antibody inhibits the interaction of zB7H6with human NKp30.
 27. The method of claim 25 or 26, wherein the antibodyis a monoclonal antibody.
 28. The method of claim 27, wherein theantibody is a human or humanized monoclonal antibody.
 29. The method ofclaim 27, wherein the antibody is a single chain antibody.
 30. A methodof screening for an antagonist of the interaction of zB7H6 with NKp30,the method comprising: (a) contacting an agent with a zB7H6 polypeptidein the presence of an NKp30 polypeptide; (b) detecting a measure of theinteraction of the zB7H6 polypeptide with the NKp30 polypeptide; (c)determining whether the level of the zB7H6/NKp30 interaction measured instep (b) is significantly less relative to the level of interactionmeasured for control zB7H6 and NKp30 polypeptides in the absence of theagent, such that if the level of zB7H6/NKp30 interaction is less, thenthe agent is identified as an antagonist of the interaction of zB7H6with NKp30.
 31. A method of screening for an agonist of the interactionof zB7H6 with NKp30, the method comprising: (a) contacting an agent witha zB7H6 polypeptide in the presence of an NKp30 polypeptide; (b)detecting a measure of the interaction of the zB7H6 polypeptide with theNKp30 polypeptide; (c) determining whether the level of the zB7H6/NKp30interaction measured in step (b) is significantly greater relative tothe level of interaction measured for control zB7H6 and NKp30polypeptides in the absence of the agent, such that if the level ofzB7H6/NKp30 interaction is greater, then the agent is identified as anagonist of the interaction of zB7H6 with NKp30.
 32. A method fordepleting or inhibiting the growth of zB7H6-expressing cells within acell population comprising said zB7H6-expressing cells, the methodcomprising: contacting said zB7H6-expressing cells with an effectiveamount of an antibody-drug conjugate comprising an antibody thatspecifically binds to a polypeptide segment consisting of the amino acidsequence set forth in residues 25-266 of SEQ ID NO:2, wherein saidantibody is conjugated to a cytotoxic agent.
 33. The method of claim 32,wherein the antibody is a monoclonal antibody.
 34. The method of claim33, wherein the antibody is a human or humanized monoclonal antibody.35. The method of claim 33, wherein the antibody is a single chainantibody.
 36. The method of claim 32, wherein the cytotoxic agent isselected from the group consisting of an anti-tubulin agent, a DNA minorgroove binding agent, a DNA minor groove alkylating agent, aduocarmycin, and a puromycin.
 37. The method of claim 36, wherein theanti-tubulin agent is selected from the group consisting of adolastatin, a vinca alkaloid, a podophyllatoxin, a taxane, a baccatinderivative, a cryptophysin, a maytansinoid, and a combretastatin. 38.The method of claim 32, wherein the antibody is conjugated to thecytotoxic agent via a linker.
 39. A method for treating azB7H6-expressing cancer in a subject, the method comprising:administering to the subject an effective amount of an antibody-drugconjugate comprising an antibody that specifically binds to apolypeptide segment consisting of the amino acid sequence set forth inresidues 25-266 of SEQ ID NO:2, wherein said antibody is conjugated to acytotoxic agent.
 40. The method of claim 39, wherein the antibody is amonoclonal antibody.
 41. The method of claim 40, wherein the antibody isa human or humanized monoclonal antibody.
 42. The method of claim 40,wherein the antibody is a single chain antibody.
 43. The method of claim39, wherein the zB7H6-expressing cancer is a cancer of the colon, liver,cervix, lung, pancreas, or prostate.
 44. The method of claim 39, whereinthe zB7H6-expressing cancer is a prohemocytic leukemia, a B-celllymphoma, a T-cell lymphoma, a monocytic lymphoma, a erythroleukemia,Burkitt's lymphoma, a chronic myelogenous leukemia, or an acutelymphoblastic leukemia.
 45. A method for inducing antibody dependentcellular cytotoxicity (ADCC) against a zB7H6-expressing cell, the methodcomprising: contacting said zB7H6-expressing cell with an effectiveamount an antibody that specifically binds to a polypeptide segmentconsisting of the amino acid sequence set forth in residues 25-266 ofSEQ ID NO:2, wherein said contacting is in the presence of an NK cell ora CD8⁺ T cell expressing an Fc receptor having ADCC activity, andwherein the antibody comprises an Fc region capable of binding said Fcreceptor.
 46. The method of claim 45, wherein the antibody is amonoclonal antibody.
 47. The method of claim 46, wherein the antibody isa human or humanized monoclonal antibody.
 48. The method of claim 46,wherein the antibody is a single chain antibody.
 49. The method of claim45, wherein the Fc region is a single chain Fc (scFc).
 50. The method ofclaim 45, wherein the zB7H6-expressing cell is a cancer cell.
 51. Themethod of claim 50, wherein the cancer cell is a colon cancer cell, aliver cancer cell, a cervical cancer cell, a lung cancer cell, apancreatic cancer cell, or a prostate cancer cell.
 52. The method ofclaim 50, wherein the cancer cell is a prohemocytic leukemia cell, aB-cell lymphoma cell, a T-cell lymphoma, a monocytic lymphoma cell, aerythroleukemia cell, a Burkitt's lymphoma cell, a chronic myelogenousleukemia cell, or an acute lymphoblastic leukemia.
 53. A method forinducing complement dependent cytotoxicity (CDC) against azB7H6-expressing cell, the method comprising: contacting saidzB7H6-expressing cell with an effective amount an antibody thatspecifically binds to a polypeptide segment consisting of the amino acidsequence set forth in residues 25-266 of SEQ ID NO:2, wherein saidcontacting is in the presence of complement, and wherein the anti-zB7H6antibody comprises an Fc region having CDC activity.
 54. The method ofclaim 53, wherein the antibody is a monoclonal antibody.
 55. The methodof claim 54, wherein the antibody is a human or humanized monoclonalantibody.
 56. The method of claim 54, wherein the antibody is a singlechain antibody.
 57. The method of claim 53, wherein the Fc region is asingle chain Fc (scFc).
 58. The method of claim 53, wherein thezB7H6-expressing cell is a cancer cell.
 59. The method of claim 58,wherein the cancer cell is a colon cancer cell, a liver cancer cell, acervical cancer cell, a lung cancer cell, a pancreatic cancer cell, or aprostate cancer cell.
 60. The method of claim 58, wherein the cancercell is a prohemocytic leukemia cell, a B-cell lymphoma cell, a T-celllymphoma, a monocytic lymphoma cell, a erythroleukemia cell, a Burkitt'slymphoma cell, a chronic myelogenous leukemia cell, or an acutelymphoblastic leukemia.
 61. A method for treating a zB7H6-expressingcancer in a subject, the method comprising: administering to the subjectan effective amount of an antibody that specifically binds to apolypeptide segment consisting of the amino acid sequence set forth inresidues 25-266 of SEQ ID NO:2, wherein the antibody comprises an Fcregion having at least one of ADCC activity and CDC activity.
 62. Themethod of claim 61, wherein the antibody is a monoclonal antibody. 63.The method of claim 62, wherein the antibody is a human or humanizedmonoclonal antibody.
 64. The method of claim 62, wherein the antibody isa single chain antibody.
 65. The method of claim 68, wherein the Fcregion is a single chain Fc (scFc).
 66. The method of claim 61, whereinthe zB7H6-expressing cancer is a cancer of the colon, liver, cervix,lung, pancreas, or prostate.
 67. The method of claim 61, wherein thezB7H6-expressing cancer is a prohemocytic leukemia, a B-cell lymphoma, aT-cell lymphoma, a monocytic lymphoma, a erythroleukemia, Burkitt'slymphoma, a chronic myelogenous leukemia, or an acute lymphoblasticleukemia.